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. 1980 Jan 1;151(1):12–19. doi: 10.1084/jem.151.1.12

Quantitative studies on the precursors of cytotoxic lymphocytes. VI. Second signal requirements of specifically activated precursors isolated 12 h after stimulation

PMCID: PMC2185751  PMID: 6965304

Abstract

It is shown that, in a mixed lymphocyte reaction, the production of cytotoxic T lymphocytes (CL) from cytotoxic T lymphocyte precursors (CLP) requires two signals which are separated time. Using a flow cytometer-cell sorter and a vital, fluorescent DNA stain, Hoechst 3342, CLP specific for the stimulator cells can be separated from other CLP and from stimulator cells 12 h after initiation of mixed lymphocyte cultures. These CLP are in a state of partial activation and can produce CL in the absence of stimulator cells if a second signal in the form of a concanavalin A-induced spleen cell supernate factor is added. Specific CL are also generated when the partially activated CLP are cultured with both nude spleen cells and stimulator cells. In this case it appears that an interaction between the stimulator cells and the nude spleen cells leads to production of the second signal.

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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. Bach F. H., Bach M. L., Sondel P. M. Differential function of major histocompatibility complex antigens in T-lymphocyte activation. Nature. 1976 Jan 29;259(5541):273–281. doi: 10.1038/259273a0. [DOI] [PubMed] [Google Scholar]
  3. Bonner W. A., Hulett H. R., Sweet R. G., Herzenberg L. A. Fluorescence activated cell sorting. Rev Sci Instrum. 1972 Mar;43(3):404–409. doi: 10.1063/1.1685647. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Heber-Katz E., Click R. E. Immune responses in vitro. V. Role of mercaptoethanol in the mixed-leukocyte reaction. Cell Immunol. 1972 Nov;5(3):410–418. doi: 10.1016/0008-8749(72)90067-6. [DOI] [PubMed] [Google Scholar]
  6. Jacobs S. W., Miller R. G. Characterization of in vitro T-lymphocyte colonies from spleens of nude mice. J Immunol. 1979 Feb;122(2):582–584. [PubMed] [Google Scholar]
  7. Julius M. H., Simpson E., Herzenberg L. A. A rapid method for the isolation of functional thymus-derived murine lymphocytes. Eur J Immunol. 1973 Oct;3(10):645–649. doi: 10.1002/eji.1830031011. [DOI] [PubMed] [Google Scholar]
  8. Lalande M. E., Miller R. G. Fluorescence flow analysis of lymphocyte activation using Hoechst 33342 dye. J Histochem Cytochem. 1979 Jan;27(1):394–397. doi: 10.1177/27.1.86569. [DOI] [PubMed] [Google Scholar]
  9. Latt S. A., Stetten G. Spectral studies on 33258 Hoechst and related bisbenzimidazole dyes useful for fluorescent detection of deoxyribonucleic acid synthesis. J Histochem Cytochem. 1976 Jan;24(1):24–33. doi: 10.1177/24.1.943439. [DOI] [PubMed] [Google Scholar]
  10. Miller R. G., Derry H. A cell population in nu/nu spleen can prevent generation of cytotoxic lymphocytes by normal spleen cells against self antigens of the nu/nu spleen. J Immunol. 1979 Apr;122(4):1502–1509. [PubMed] [Google Scholar]
  11. Miller R. G., Dunkley M. Quantitative analysis of the 51Cr release cytotoxicity assay for cytotoxic lymphocytes. Cell Immunol. 1974 Nov;14(2):284–302. doi: 10.1016/0008-8749(74)90212-3. [DOI] [PubMed] [Google Scholar]
  12. Miller R. G., Teh H. S., Harley E., Phillips R. A. Quantitative studies of the activation of cytotoxic lymphocyte precursor cells. Immunol Rev. 1977;35:38–58. doi: 10.1111/j.1600-065x.1977.tb00234.x. [DOI] [PubMed] [Google Scholar]
  13. Okada M., Klimpel G. R., Kuppers R. C., Henney C. S. The differentiation of cytotoxic T cells in vitro. I. Amplifying factor(s) in the primary response is Lyt 1 + cell dependent. J Immunol. 1979 Jun;122(6):2527–2533. [PubMed] [Google Scholar]
  14. Paetkau V., Mills G., Gerhart S., Monticone V. Proliferation of murine thymic lymphocytes in vitro is mediated by the concanavalin A-induced release of a lymphokine (costimulator). J Immunol. 1976 Oct;117(4):1320–1324. [PubMed] [Google Scholar]
  15. Pallavicini M. G., Lalande M. E., Miller R. G., Hill R. P. Cell cycle distribution of chronically hypoxic cells and determination of the clonogenic potential of cells accumulated in G2 + M phases after irradiation of a solid tumor in vivo. Cancer Res. 1979 Jun;39(6 Pt 1):1891–1897. [PubMed] [Google Scholar]
  16. 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]
  17. Price G. B., McCutcheon M. J., Taylor W. B., Miller R. G. Measurement of cytoplasmic fluorescence depolarization of single cells in a flow system. J Histochem Cytochem. 1977 Jul;25(7):597–600. doi: 10.1177/25.7.302266. [DOI] [PubMed] [Google Scholar]
  18. Talmage D. W., Woolnough J. A., Hemmingsen H., Lopez L., Lafferty K. J. Activation of cytotoxic T cells by nonstimulating tumor cells and spleen cell factor(s). Proc Natl Acad Sci U S A. 1977 Oct;74(10):4610–4614. doi: 10.1073/pnas.74.10.4610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Teh H. S., Phillips R. A., Miller R. G. Quantitative studies on the precursors of cytotoxic lymphocytes. II. Specificity of precursors responsive to alloantigens and to concanavalin A. J Immunol. 1977 Mar;118(3):1057–1062. [PubMed] [Google Scholar]
  20. Teh H. S., Phillips R. A., Miller R. G. Quantitative studies on the precursors of cytotoxic lymphocytes. V. The cellular basis for the cross-reactivity of TNP-specific clones. J Immunol. 1978 Nov;121(5):1711–1717. [PubMed] [Google Scholar]
  21. Watson J., Aarden L. A., Shaw J., Paetkau V. Molecular and quantitative analysis of helper T cell-replacing factors on the induction of antigen-sensitive B and T lymphocytes. J Immunol. 1979 May;122(5):1633–1638. [PubMed] [Google Scholar]

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