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. 1982 Feb 1;155(2):358–379. doi: 10.1084/jem.155.2.358

Phenotypic and functional properties of murine thymocytes. I. Precursors of cytolytic T lymphocytes and interleukin 2-producing cells are all contained within a subpopulation of “mature” thymocytes as analyzed by monoclonal antibodies and flow microfluorometry

R Ceredig, AL Glasebrook, HR MacDonald
PMCID: PMC2186593  PMID: 6120202

Abstract

The correlation between surface phenotype and function in subpopulations of murine thymocytes has been investigated using flow microfluorometry (FMF). C57BL/6 thymocytes stained with monoclonal antibodies directed against Lyt-2, H-2K(b), and Thy-l.2 and passed on an FACS II flow cytometer could be resolved into at least four distinct subpopulations on the basis of fluorescence and forward light scatter (FLS) measurements. (a) Medium-sized Lyt-2(+) cells that stained strongly with H-2K(b) and weakly with Thy-l.2 (5 percent of total cells); (b) medium-sized Lyt-2(-) cells with other properties as in (a) (10 percent); (c) small Lyt-2(+) cells that stained weakly with H-2K(b) and strongly with Thy-l.2 (60 percent); and (d) large Lyt-2(+) cells that stained weakly with H-2K(b) and very strongly with Thy- 1.2 (23 percent). Cortisone-resistant thymocytes (CRT) were found to correspond phenotypically to populations (a) and (b). The distribution of cytolytic T lymphocyte precursors (CTL-P) directed against H-2(d) alloantigens in subpopulations of C57BL/6 thymocytes that had been sorted according to the phenotypic criteria described above was then investigated. CTL-P in sorted and control populations were quantitated by limiting dilution analysis of mixed leukocyte microcultures established in an excess of interleukin 2 (IL-2). These studies established that all thymus CTL-P could be quantitatively recovered in a subpopulation of cells that was cortisone-resistant, medium-sized, Lyt-2(+), H-2K(b+), and weakly stained with Thy-l.2. In parallel studies, the production of IL-2 by subpopulations of C57BL/6 thymocytes was quantitatively assessed using a recently developed sensitive microassay system. Graded numbers of sorted or control thymocytes were stimulated with irradiated T cell-depleted allogeneic cells and assayed for their ability to support the growth of an IL-2-dependent cytolytic T lymphocyte clone. Using this method, IL-2 production was found to reside entirely in a subpopulation of cortisone-resistant, medium-sized Lyt-2(-) thymocytes. Further phenotypic analysis of this subpopulation of cells indicated that it was homogeneously H-2K(b+) and weakly staining with Thy- 1.2. Taken together with the CTL-P results, these data directly demonstrate that a subpopulation of thymocytes with a mature phenotype (i.e., cortisone- resistant, medium-sized, H-2K(b+), and weakly staining with Thy-l.2) accounts for all the functional activity in the thymus. Reasons for the apparent discrepancy between these results and other recent studies will be discussed.

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Selected References

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  1. Blomgren H., Andersson B. Characteristics of the immunocompetent cells in the mouse thymus: cell population changes during cortisone-induced atrophy and subsequent regeneration. Cell Immunol. 1970 Nov;1(5):545–560. doi: 10.1016/0008-8749(70)90041-9. [DOI] [PubMed] [Google Scholar]
  2. Cantor H., Asofsky R. Synergy among lymphoid cells mediating the graft-versus-host response. 3. Evidence for interaction between two types of thymus-derived cells. J Exp Med. 1972 Apr 1;135(4):764–779. doi: 10.1084/jem.135.4.764. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cantor H., Boyse E. A. Functional subclasses of T lymphocytes bearing different Ly antigens. II. Cooperation between subclasses of Ly+ cells in the generation of killer activity. J Exp Med. 1975 Jun 1;141(6):1390–1399. doi: 10.1084/jem.141.6.1390. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cerottini J. C., Brunner K. T. Localization of mouse isoantigens on the cell surface as revealed by immunofluorescence. Immunology. 1967 Oct;13(4):395–403. [PMC free article] [PubMed] [Google Scholar]
  5. Cerottini J. C., Engers H. D., Macdonald H. R., Brunner T. Generation of cytotoxic T lymphocytes in vitro. I. Response of normal and immune mouse spleen cells in mixed leukocyte cultures. J Exp Med. 1974 Sep 1;140(3):703–717. doi: 10.1084/jem.140.3.703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cerottini J. C., MacDonald H. R. Limiting dilution analysis of alloantigen-reactive T lymphocytes. V. Lyt phenotype of cytolytic T lymphocyte precursors reactive against normal and mutant H-2 antigens. J Immunol. 1981 Feb;126(2):490–496. [PubMed] [Google Scholar]
  7. Droege W., Zucker R. Lymphocyte subpopulations in the thymus. Transplant Rev. 1975;25:3–25. doi: 10.1111/j.1600-065x.1975.tb00724.x. [DOI] [PubMed] [Google Scholar]
  8. Emerson S. G., Murphy D. B., Cone R. E. Selective turnover and shedding of H-2K and H-2D antigens is controlled by the major histocompatibility complex. Implications for H-2-restricted recognition. J Exp Med. 1980 Oct 1;152(4):783–795. doi: 10.1084/jem.152.4.783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fathman C. G., Small M., Herzenberg L. A., Weissman I. L. Thymus cell maturation. II. Differentiation of three "mature" subclasses in vivo. Cell Immunol. 1975 Jan;15(1):109–128. doi: 10.1016/0008-8749(75)90169-0. [DOI] [PubMed] [Google Scholar]
  10. Fowlkes B. J., Waxdal M. J., Sharrow S. O., Thomas C. A., 3rd, Asofsky R., Mathieson B. J. Differential binding of fluorescein-labeled lectins to mouse thymocytes: subsets revealed by flow microfluorometry. J Immunol. 1980 Aug;125(2):623–630. [PubMed] [Google Scholar]
  11. Gillis S., Ferm M. M., Ou W., Smith K. A. T cell growth factor: parameters of production and a quantitative microassay for activity. J Immunol. 1978 Jun;120(6):2027–2032. [PubMed] [Google Scholar]
  12. Glasebrook A. L., Fitch F. W. T-cell lines which cooperate in generation of specific cytolytic activity. Nature. 1979 Mar 8;278(5700):171–173. doi: 10.1038/278171a0. [DOI] [PubMed] [Google Scholar]
  13. Goding J. W. Antibody production by hybridomas. J Immunol Methods. 1980;39(4):285–308. doi: 10.1016/0022-1759(80)90230-6. [DOI] [PubMed] [Google Scholar]
  14. Irlé C., Piguet P. F., Vassalli P. In vitro maturation of immature thymocytes into immunocompetent T cells in the absence of direct thymic influence. J Exp Med. 1978 Jul 1;148(1):32–45. doi: 10.1084/jem.148.1.32. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kruisbeek A. M., Zijlstra J. J., Kröse T. J. Distinct effects of T cell growth factors and thymic epithelial factors on the generation of cytotoxic T lymphocytes by thymocyte subpopulations. J Immunol. 1980 Sep;125(3):995–1002. [PubMed] [Google Scholar]
  16. Ledbetter J. A., Herzenberg L. A. Xenogeneic monoclonal antibodies to mouse lymphoid differentiation antigens. Immunol Rev. 1979;47:63–90. doi: 10.1111/j.1600-065x.1979.tb00289.x. [DOI] [PubMed] [Google Scholar]
  17. Lutz C. T. The generation of cytolytic activity in mixed leukocyte cultures: cortisone-resistant thymocytes are deficient in helper T lymphocytes. J Immunol. 1981 Sep;127(3):1156–1163. [PubMed] [Google Scholar]
  18. MacDonald H. R., Cerottini J. C., Ryser J. E., Maryanski J. L., Taswell C., Widmer M. B., Brunner K. T. Quantitation and cloning of cytolytic T lymphocytes and their precursors. Immunol Rev. 1980;51:93–123. doi: 10.1111/j.1600-065x.1980.tb00318.x. [DOI] [PubMed] [Google Scholar]
  19. MacDonald H. R., Phillips R. A., Miller R. G. Allograft immunity in the mouse. II. Physical studies of the development of cytotoxic effector cells from their immediate progenitors. J Immunol. 1973 Aug;111(2):575–589. [PubMed] [Google Scholar]
  20. Mathieson B. J., Sharrow S. O., Campbell P. S., Asofsky R. An Lyt differentiated thymocyte subpopulation detected by flow microfluorometry. Nature. 1979 Feb 8;277(5696):478–480. doi: 10.1038/277478a0. [DOI] [PubMed] [Google Scholar]
  21. Miller J. F., Osoba D. Current concepts of the immunological function of the thymus. Physiol Rev. 1967 Jul;47(3):437–520. doi: 10.1152/physrev.1967.47.3.437. [DOI] [PubMed] [Google Scholar]
  22. Pilarski L. M. A requirement for antigen-specific helper T cells in the generation of cytotoxic T cells from thymocyte precursors. J Exp Med. 1977 Mar 1;145(3):709–725. doi: 10.1084/jem.145.3.709. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Reisner Y., Linker-Israeli M., Sharon N. Separation of mouse thymocytes into two subpopulations by the use of peanut agglutinin. Cell Immunol. 1976 Jul;25(1):129–134. doi: 10.1016/0008-8749(76)90103-9. [DOI] [PubMed] [Google Scholar]
  24. Roelants G. E., London J., Mayor-Withey K. S., Serrano B. Peanut agglutinin. II. Characterization of the Thy-1, Tla and Ig phenotype of peanut agglutinin-positive cells in adult, embryonic and nude mice using double immunofluorescence. Eur J Immunol. 1979 Feb;9(2):139–145. doi: 10.1002/eji.1830090209. [DOI] [PubMed] [Google Scholar]
  25. Ryser J. E., Cerottini J. C., Brunner K. T. Generation of cytolytic T lymphocytes in vitro. IX. induction of secondary CTL responses in primary long-term MLC by supernatants from secondary MLC. J Immunol. 1978 Feb;120(2):370–377. [PubMed] [Google Scholar]
  26. Ryser J. E., MacDonald H. R. Limiting dilution analysis of alloantigen-reactive T lymphocytes. I. Comparison of precursor frequencies for proliferative and cytolytic responses. J Immunol. 1979 May;122(5):1691–1696. [PubMed] [Google Scholar]
  27. Sarmiento M., Glasebrook A. L., Fitch F. W. IgG or IgM monoclonal antibodies reactive with different determinants on the molecular complex bearing Lyt 2 antigen block T cell-mediated cytolysis in the absence of complement. J Immunol. 1980 Dec;125(6):2665–2672. [PubMed] [Google Scholar]
  28. Scollay R., Jacobs S., Jerabek L., Butcher E., Weissman I. T cell maturation: thymocyte and thymus migrant subpopulations defined with monoclonal antibodies to MHC region antigens. J Immunol. 1980 Jun;124(6):2845–2853. [PubMed] [Google Scholar]
  29. Shortman K., Linthicum D. S., Battye F. L., Goldschneider I., Liabeuf A., Golstein P., Clark E. A., Lake P. Cytotoxic and fluorescent assays for thymocyte subpopulations differing in surface thy-1 level. Cell Biophys. 1979 Sep;1(3):255–270. doi: 10.1007/BF02783667. [DOI] [PubMed] [Google Scholar]
  30. Shortman K., Von Boehmer H., Lipp J., Hopper K. Subpopulations of T-lymphocytes. Physical separation, functional specialisation and differentiation pathways of sub-sets of thymocytes and thymus-dependent peripheral lymphocytes. Transplant Rev. 1975;25:163–210. [PubMed] [Google Scholar]
  31. 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]
  32. Swain S. L. Association of Ly phenotypes, T cell function and MHC recognition. Fed Proc. 1980 Nov;39(13):3110–3113. [PubMed] [Google Scholar]
  33. Tartakovsky B., De Baetselier P., Segal S. Serological detection of H-2K- and H-2D-gene products. I. Principal difference between T and B lymphocytes in expression of H-2D-encoded alloantigens. Immunogenetics. 1980;11(6):585–595. doi: 10.1007/BF01567827. [DOI] [PubMed] [Google Scholar]
  34. Taswell C. Limiting dilution assays for the determination of immunocompetent cell frequencies. I. Data analysis. J Immunol. 1981 Apr;126(4):1614–1619. [PubMed] [Google Scholar]
  35. Taswell C., MacDonald H. R., Cerottini J. C. Limiting dilution analysis of alloantigen-reactive T lymphocytes. II. Effect of cortisone and cyclophosphamide on cytolytic T lymphocyte precursor frequencies in the thymus. Thymus. 1979 Sep;1(1-2):119–131. [PubMed] [Google Scholar]
  36. Teh H. S., Teh S. J. The use of a clonal assay for cytotoxic T lymphocytes to determine the Ly phenotypes of the cytotoxic precursor and effector cells to alloantigens and trinitrophenyl-modified self antigens. J Immunol. 1980 Nov;125(5):1977–1986. [PubMed] [Google Scholar]
  37. Wagner H., Hardt C., Bartlett R., Röllinghoff M., Pfizenmaier K. Intrathymic differentiation of cytotoxic T lymphocyte (CTL) precursors. I. The CTL immunocompetence of peanut agglutinin-positive (cortical) and negative (medullary) Lyt 123 thymocytes. J Immunol. 1980 Dec;125(6):2532–2538. [PubMed] [Google Scholar]
  38. Wagner H., Harris A. W., Feldmann M. Cell-mediated immune response in vitro. II. The role of thymus and thymus-derived lymphocytes. Cell Immunol. 1972 May;4(1):39–50. doi: 10.1016/0008-8749(72)90004-4. [DOI] [PubMed] [Google Scholar]
  39. Wagner H., Röllinghoff M. T-T-cell interactions during the vitro cytotoxic allograft responses. I. Soluble products from activated Lyl+ T cells trigger autonomously antigen-primed Ly23+ T cells to cell proliferation and cytolytic activity. J Exp Med. 1978 Dec 1;148(6):1523–1538. doi: 10.1084/jem.148.6.1523. [DOI] [PMC free article] [PubMed] [Google Scholar]

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