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. 1970 Jul 1;132(1):16–30. doi: 10.1084/jem.132.1.16

A PLAQUE ASSAY FOR ENUMERATING ANTIGEN-SENSITIVE CELLS IN DELAYED-TYPE HYPERSENSITIVITY

Barry R Bloom 1, Luis Jimenez 1, Philip I Marcus 1
PMCID: PMC2138748  PMID: 4323747

Abstract

A general method is described for enumerating antigen-sensitive lymphocytes obtained from individuals having delayed hypersensitivity, in this case from highly tuberculin-sensitive guinea pigs. The method is based on the observation that resting lymphocytes are generally unable to support replication of RNA viruses, whereas antigen-"activated" lymphocytes can. Lymph node lymphocytes from individual animals were cultured in the presence or absence of tuberculin purified protein derivatives (PPD). After various periods of time, the cells were infected either with Newcastle disease virus or vesicular stomatitis virus, and plated in agar over a monolayer of cells susceptible to the virus. Wherever a lymphocyte yielded infectious virus, a discrete plaque in the monolayer could be seen. The increase in plaques of the antigen-stimulated cells over the background of the control sample was taken as the number of antigen-sensitive cells in the population. When lymphocytes from normal guinea pigs or from guinea pigs immunized to produce only circulating antibody to PPD were similarly tested, no increase in plaque-forming units (PFU) was observed. The average increase in PFU due to antigenic stimulation varied from 1 per 1000 lymphocytes at 24 hr to 16 per 1000 lymphocytes at 96 hr. By employing inhibitors of mitosis (colchicine, vinblastine, and thymidine) it was evident that the increase in PFU at least up to 48 hr was primarily due to initial antigen-reactive cells and not their progeny.

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Selected References

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

  1. BERG R. B. Multiplication of ECHO 9 virus in suspensions of human leucocytes. Proc Soc Exp Biol Med. 1961 Dec;108:742–744. [PubMed] [Google Scholar]
  2. Bach F. H., Bock H., Graupner K., Day E., Klostermann H. Cell kinetic studies in mixed leukocyte cultures: an in vitro model of homograft reactivity. Proc Natl Acad Sci U S A. 1969 Feb;62(2):377–384. doi: 10.1073/pnas.62.2.377. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bennett B., Bloom B. R. Reactions in vivo and in vitro produced by a soluble substance associated with delayed-type hypersensitivity. Proc Natl Acad Sci U S A. 1968 Mar;59(3):756–762. doi: 10.1073/pnas.59.3.756. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bennett B., Bloom B. R. Studies on the migration inhibitory factor associated with delayed-type hypersensitivity: cytodynamics and specificity. Transplantation. 1967 Jul;5(4 Suppl):996–1000. [PubMed] [Google Scholar]
  5. Bloom B. R., Bennett B. Migration inhibitory factor associated with delayed-type hypersensitivity. Fed Proc. 1968 Jan-Feb;27(1):13–15. [PubMed] [Google Scholar]
  6. David J. R. Macrophage migration. Fed Proc. 1968 Jan-Feb;27(1):6–12. [PubMed] [Google Scholar]
  7. Edelman R., Wheelock E. F. Specific role of each human leukocyte type in viral infections. I. Monocyte as host cell for vesicular stomatitis virus replication in vitro. J Virol. 1967 Dec;1(6):1139–1149. doi: 10.1128/jvi.1.6.1139-1149.1967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Edelman R., Wheelock E. F. Vesicular stomatitis virus replication in human leukocyte cultures: enhancement by phytohemagglutinin. Science. 1966 Nov 25;154(3752):1053–1055. doi: 10.1126/science.154.3752.1053. [DOI] [PubMed] [Google Scholar]
  9. Fox A. E., Anschel J., Evans G. L., Mohan R. R., Schwartz B. S. Isolation of a Soluble Resistance-Enhancing Factor from Mycobacterium phlei. J Bacteriol. 1966 Aug;92(2):285–290. doi: 10.1128/jb.92.2.285-290.1966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Granger G. A., Shacks S. J., Williams T. W., Kolb W. P. Lymphocyte in vitro cytotoxicity: specific release of lymphotoxin-like materials from tuberculin-sensitive lymphoid cells. Nature. 1969 Mar 22;221(5186):1155–1157. doi: 10.1038/2211155a0. [DOI] [PubMed] [Google Scholar]
  11. Green J. A., Cooperband S. R., Kibrick S. Immune specific induction of interferon production in cultures of human blood lymphocytes. Science. 1969 Jun 20;164(3886):1415–1417. doi: 10.1126/science.164.3886.1415. [DOI] [PubMed] [Google Scholar]
  12. Gresser I., Lang D. J. Relationships between viruses and leucocytes. Prog Med Virol. 1966;8:62–130. [PubMed] [Google Scholar]
  13. Heise E. R., Han S., Weiser R. S. In vitro studies on the mechanism of macrophage migration inhibition in tuberculin sensitivity. J Immunol. 1968 Nov;101(5):1004–1015. [PubMed] [Google Scholar]
  14. Krejcí J., Pekárek J., Johanovský J., Svejcar J. Demonstration of the inflammatory activity of the supernatant of hypersensitive lymph node cells incubated with a high dose of antigen. Immunology. 1969 May;16(5):677–684. [PMC free article] [PubMed] [Google Scholar]
  15. MARCUS P. I., PUCK T. T. Host-cell interaction of animal viruses. I. Titration of cell-killing by viruses. Virology. 1958 Oct;6(2):405–423. doi: 10.1016/0042-6822(58)90091-6. [DOI] [PubMed] [Google Scholar]
  16. Newsome J. Synthesis of ribonucleic acid by stimulated human lymphocytes. Nature. 1965 Jun 5;206(988):1013–1015. doi: 10.1038/2061013a0. [DOI] [PubMed] [Google Scholar]
  17. Perlmann P., Holm G. Cytotoxic effects of lymphoid cells in vitro. Adv Immunol. 1969;11:117–193. doi: 10.1016/s0065-2776(08)60479-4. [DOI] [PubMed] [Google Scholar]
  18. Valentine F. T., Lawrence H. S. Lymphocyte stimulation: transfer of cellular hypersensitivity to antigen in vitro. Science. 1969 Sep 5;165(3897):1014–1016. doi: 10.1126/science.165.3897.1014. [DOI] [PubMed] [Google Scholar]
  19. Ward P. A., Remold H. G., David J. R. Leukotactic factor produced by sensitized lymphocytes. Science. 1969 Mar 7;163(3871):1079–1081. doi: 10.1126/science.163.3871.1079. [DOI] [PubMed] [Google Scholar]
  20. Wheelock E. F. Virus replication and high-titered interferon production in human leukocyte cultures inoculated with Newcastle disease virus. J Bacteriol. 1966 Nov;92(5):1415–1421. doi: 10.1128/jb.92.5.1415-1421.1966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Willems F. T., Melnick J. L., Rawls W. E. Replication of poliovirus in phytohemagglutinin-stimulated human lymphocytes. J Virol. 1969 May;3(5):451–457. doi: 10.1128/jvi.3.5.451-457.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Wilson D. B., Blyth JL NOWELL P. C. Quantitative studies on the mixed lymphocyte interaction in rats. 3. Kinetics of the response. J Exp Med. 1968 Nov 1;128(5):1157–1181. doi: 10.1084/jem.128.5.1157. [DOI] [PMC free article] [PubMed] [Google Scholar]

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