Skip to main content
PMC home page
The Journal of Experimental Medicine logoLink to The Journal of Experimental Medicine
. 1978 Feb 1;147(2):297–315. doi: 10.1084/jem.147.2.297

Mouse spleen lymphoblasts generated in vitro. Their replication and differentiation in vitro

RM Steinman, BG Machtinger, J Fried, ZA Cohn
PMCID: PMC2184482  PMID: 564390

Abstract

Mouse spleen lymphoblasts induced with lipopolysaccharide and fetal calf serum were obtained in high yield and purity in their first proliferative cell cycle by floatation in dense bovine plasma albumin columns (3). The blasts were maintained in vitro for 3 more days. The cultures were examined in bulk on each day, and in addition, those cells in S phase initially were tagged with [(3)H]thymidine and followed continuously in vitro. Grain count dilution data indicated that most blasts divided but twice over a 2- to 3-day interval in vitro. [(3)H]Thymidine pulse radiolabeling and flow microfluorometry suggested that at least 50-70 percent of the proliferating blasts withdrew from proliferative activity after 2-3 days of culture. Morphologic studies demonstrated that lymphoblasts persisted as such for 1-2 days in vitro and then matured into typical plasma cells. Many of the blastprogeny had small nuclei and considerable basophilic cytoplasm on Giemsa-stained cell smears; abundant rough endoplasmic reticulum by electron microscopy; and readily detectable cytoplasmic Ig by immunocytochemistry. Reversion of blasts to small lymphocytes could not be detected; however, some blasts persisted even after 3 days of culture. The viability of the cultured lymphoblast was followed by initially tagging the cells with [(3)H]thymidine as well as several other techniques. Little cell death was documented during the first day of culture. The number of labeled progeny increased twofold whereas the grain count halved. But 40- 50 percent of the cell-associated label was lost during each of the second and third days, and fewer labeled progeny than predicted by grain count dilution were identified. The culture medium could not be implicated in this loss of lymphoblast progeny, and we suggest that the maturation of the lymphoblast to a short-lived plasma cell was responsible. Therefore mitogen-stimulated B blasts seem to mature into typical plasma cells after just two cycles of cell division. The plasma cells resemble those produced in situ during an immune response in their cytologic features, withdrawal from active proliferative activity, and short life-span.

Full Text

The Full Text of this article is available as a PDF (2.5 MB).

Selected References

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

  1. Andersson J., Coutinho A., Lernhardt W., Melchers F. Clonal growth and maturation to immunoglobulin secretion in vitro of every growth-inducible B lymphocyte. Cell. 1977 Jan;10(1):27–34. doi: 10.1016/0092-8674(77)90136-2. [DOI] [PubMed] [Google Scholar]
  2. Andersson J., Melchers F. Maturation of mitogen-activated bone marrow-derived lymphocytes in the absence of proliferation. Eur J Immunol. 1974 Aug;4(8):533–539. doi: 10.1002/eji.1830040803. [DOI] [PubMed] [Google Scholar]
  3. Andersson J., Möller G., Sjöberg O. Selective induction of DNA synthesis in T and B lymphocytes. Cell Immunol. 1972 Aug;4(4):381–393. doi: 10.1016/0008-8749(72)90040-8. [DOI] [PubMed] [Google Scholar]
  4. Andersson L. C., Häyry P. Generation of T memory cells in one-way mixed lymphocyte culture. I. Selective recovery of activated cells and their reversion to 'secondary' lymphocytes. Scand J Immunol. 1974;3(4):461–469. doi: 10.1111/j.1365-3083.1974.tb01279.x. [DOI] [PubMed] [Google Scholar]
  5. Askonas B. A., Williamson A. R. Factors affecting the propagation of a B cell clone forming antibody to the 2,4-dinitrophenyl group. Eur J Immunol. 1972 Dec;2(6):487–493. doi: 10.1002/eji.1830020603. [DOI] [PubMed] [Google Scholar]
  6. Avrameas S., Leduc E. H. Detection of simultaneous antibody synthesis in plasma cells and specialized lymphocytes in rabbit lymph nodes. J Exp Med. 1970 Jun 1;131(6):1137–1168. doi: 10.1084/jem.131.6.1137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Avrameas S., Ternynck T. Peroxidase labelled antibody and Fab conjugates with enhanced intracellular penetration. Immunochemistry. 1971 Dec;8(12):1175–1179. doi: 10.1016/0019-2791(71)90395-8. [DOI] [PubMed] [Google Scholar]
  8. BALFOUR B. M., COOPER E. H., ALPEN E. L. MORPHOLOGICAL AND KINETIC STUDIES ON ANTIBODY-PRODUCING CELLS IN RAT LYMPH NODES. Immunology. 1965 Mar;8:230–244. [PMC free article] [PubMed] [Google Scholar]
  9. BESSIS M. C. Ultrastructure of lymphoid and plasma cells in realtion to globulin and antibody formation. Lab Invest. 1961 Nov-Dec;10:1040–1067. [PubMed] [Google Scholar]
  10. Bosma M., Weiler E. The clonal nature of antibody formation. I. Clones of antibody-forming cells of poly-D-alanine specificity. J Immunol. 1970 Jan;104(1):203–214. [PubMed] [Google Scholar]
  11. COOPER E. H. Production of lymphocytes and plasma cells in the rat following immunization with human serum albumin. Immunology. 1961 Jul;4:219–231. [PMC free article] [PubMed] [Google Scholar]
  12. Fried J. Analysis of deoxyribonucleic acid histograms from flow cytofluorometry. Estimation of the distribution of cells within S phase. J Histochem Cytochem. 1977 Jul;25(7):942–951. doi: 10.1177/25.7.894010. [DOI] [PubMed] [Google Scholar]
  13. Fried J. Method for the quantitative evaluation of data from flow microfluorometry. Comput Biomed Res. 1976 Jun;9(3):263–276. doi: 10.1016/0010-4809(76)90006-9. [DOI] [PubMed] [Google Scholar]
  14. Fried J., Yataganas X., Kitahara T., Perez A., Ferguson R., Sullivan S., Clarkson B. Quantitative analysis of flow microfluorometric data from asynchronous and drug-treated cell populations. Comput Biomed Res. 1976 Jun;9(3):277–290. doi: 10.1016/0010-4809(76)90007-0. [DOI] [PubMed] [Google Scholar]
  15. Graham R. C., Jr, Karnovsky M. J. The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. J Histochem Cytochem. 1966 Apr;14(4):291–302. doi: 10.1177/14.4.291. [DOI] [PubMed] [Google Scholar]
  16. Harris T. N., Hummeler K., Harris S. Electron microscopic observations on antibody-producing lymph node cells. J Exp Med. 1966 Jan 1;123(1):161–172. doi: 10.1084/jem.123.1.161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kennedy J. C., Till J. E., Siminovitch L., McCulloch E. A. The proliferative capacity of antigen-sensitive precursors of hemolytic plaque-forming cells. J Immunol. 1966 Jun;96(6):973–980. [PubMed] [Google Scholar]
  18. LEDUC E. H., COONS A. H., CONNOLLY J. M. Studies on antibody production. II. The primary and secondary responses in the popliteal lymph node of the rabbit. J Exp Med. 1955 Jul 1;102(1):61–72. doi: 10.1084/jem.102.1.61. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lindahl K. F., Wilson D. B. Histocompatibility antigen-activated cytotoxic T lymphocytes. II. Estimates of the frequency and specificity of precursors. J Exp Med. 1977 Mar 1;145(3):508–522. doi: 10.1084/jem.145.3.508. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. MOVAT H. Z., FERNANDO N. V. THE FINE STRUCTURE OF THE LYMPHOID TISSUE DURING ANTIBODY FORMATION. Exp Mol Pathol. 1965 Apr;28:155–188. doi: 10.1016/0014-4800(65)90031-6. [DOI] [PubMed] [Google Scholar]
  21. Marbrook J., Haskill J. S. The in vitro response to sheep erythrocytes by mouse spleen cells: segregation of distinct events leading to antibody formation. Cell Immunol. 1974 Jul;13(1):12–21. doi: 10.1016/0008-8749(74)90222-6. [DOI] [PubMed] [Google Scholar]
  22. Metcalf D., Nossal G. J., Warner N. L., Miller J. F., Mandel T. E., Layton J. E., Gutman G. A. Growth of B-lymphocyte colonies in vitro. J Exp Med. 1975 Dec 1;142(6):1534–1549. doi: 10.1084/jem.142.6.1534. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. NOSSAL G. J., MAKELA O. Autoradiographic studies on the immune response.I. The kinetics of plasma cell proliferation. J Exp Med. 1962 Jan 1;115:209–230. doi: 10.1084/jem.115.1.209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Nakane P. K., Kawaoi A. Peroxidase-labeled antibody. A new method of conjugation. J Histochem Cytochem. 1974 Dec;22(12):1084–1091. doi: 10.1177/22.12.1084. [DOI] [PubMed] [Google Scholar]
  25. RIEKE W. O. The in vivo reutilization of lymphocytic and sarcoma DNA by cells growing in the peritoneal cavity. J Cell Biol. 1962 May;13:205–216. doi: 10.1083/jcb.13.2.205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. SCHOOLEY J. C. Autoradiographic observations of plasma cell formation. J Immunol. 1961 Mar;86:331–337. [PubMed] [Google Scholar]
  27. Steinman R. M., Machtinger B. G., Fried J., Cohn Z. A. Mouse spleen lymphoblasts generated in vitro. Recovery in high yield and purity after floatation in dense bovine plasma albumin solutions. J Exp Med. 1978 Feb 1;147(2):279–296. doi: 10.1084/jem.147.2.279. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Stewart C. C., Cramer S. F., Steward P. G. The response of human peripheral blood lymphocytes to phytohemagglutinin: determination of cell numbers. Cell Immunol. 1975 Apr;16(2):237–250. doi: 10.1016/0008-8749(75)90115-x. [DOI] [PubMed] [Google Scholar]
  29. Teh H. S., Harley E., Phillips R. A., Miller R. G. Quantitative studies on the precursors of cytotoxic lymphocytes. I. Characterization of a clonal assay and determination of the size of clones derived from single precursors. J Immunol. 1977 Mar;118(3):1049–1056. [PubMed] [Google Scholar]
  30. Zagury D., Bernard J., Lemieux S., Mazie J. C., Avrameas S., Bussard A. E. The relationship between storage and secretion of specific antibody by immune lymphoid cells: ultrastructural localization of anti-peroxidase antibodies in plaque-forming cells of the rabbit popliteal lymph node. Eur J Immunol. 1976 Mar;6(3):194–199. doi: 10.1002/eji.1830060310. [DOI] [PubMed] [Google Scholar]
  31. Zauderer M., Askonas B. A. Several proliferative phases precede maturation of IgG-secreting cells in mitogen-stimulated cultures. Nature. 1976 Apr 15;260(5552):611–613. doi: 10.1038/260611a0. [DOI] [PubMed] [Google Scholar]

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

RESOURCES