Skip to main content
PMC home page
The Journal of Experimental Medicine logoLink to The Journal of Experimental Medicine
. 1982 Dec 1;156(6):1635–1649. doi: 10.1084/jem.156.6.1635

Definition of conditions that enable antigen-specific activation of the majority of isolated trinitrophenol-binding B cells

PMCID: PMC2186872  PMID: 6184434

Abstract

In an effort to further elucidate the early cellular events in generation of antibody responses, we have determined the requirements for antigen-specific initiation of the G0 to G1 transition by isolated trinitrophenol (TNP) -binding B lymphocytes. TNP-binding cells were isolated from normal B6D2F1 splenocyte populations using hapten affinity fractionation on disulfide-bonded TNP-gelatin-coated plates. Populations prepared in this way are greater than or equal to 96% immunoglobulin positive and 70-95% antigen binding. Isolated cells were cultured for 48 h in the presence of a variety of TNP conjugates including TNP-Brucella abortus (Ba), TNP-Ficoll, TNP-sheep erythrocytes (SRBC), TNP-human gamma globulin (HGG), or TNP-ovalbumin (OVA) before being harvested and subjected to acridine orange cell cycle analysis. As many as 80% of cells were in cycle by 48 h in response to TNP-Ba, a thymus-independent (TI1 antigen. A smaller proportion (congruent to 40%) were in cycle in response to TNP-Ficoll, a TI2 antigen. Significant activation was not detected in cultures challenged with the thymus-dependent immunogens TNP-SRBC, TNP-HGG, and TNP-OVA. Addition of interleukin 1 (IL-1), IL-2, B cell growth factor, and/or T cell- replacing factor to cultures did not facilitate responses to these immunogens, suggesting a requirement for antigen-specific T cell help for entry into cell cycle induced by thymus dependent antigens. Activation by TNP-Ba was antigen specific and independent of accessory cells, occurring with equal efficiency in bulk and single-cell cultures. Activation by TNP-Ba was inhibitable by anti-Fab and anti-mu antibodies, but not by anti-delta antibodies. Results indicate that activation of TNP-binding cells to enter cell cycle by TNP-Ba is independent of accessory cells and requires interaction of antigen with cell surface IgM. Exposure to thymus-dependent TNP-immunogens plus nonspecific helper factors is insufficient to cause entry of TNP- binding cells into cycle.

Full Text

The Full Text of this article is available as a PDF (1.2 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., Melchers F. Frequencies of mitogen-reactive B cells in the mouse. I. Distribution in different lymphoid organs from different inbred strains of mice at different ages. J Exp Med. 1977 Jun 1;145(6):1511–1519. doi: 10.1084/jem.145.6.1511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Andersson J., Schreier M. H., Melchers F. T-cell-dependent B-cell stimulation is H-2 restricted and antigen dependent only at the resting B-cell level. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1612–1616. doi: 10.1073/pnas.77.3.1612. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Andres C. M., Maddalena A., Hudak S., Young N. M., Claflin J. L. Anti-phosphocholine hybridoma antibodies. II. Functional analysis of binding sites within three antibody families. J Exp Med. 1981 Nov 1;154(5):1584–1598. doi: 10.1084/jem.154.5.1584. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bach J. F., Muller J. Y., Dardenne M. In vivo specific antigen recognition by rosette forming cells. Nature. 1970 Sep 19;227(5264):1251–1252. doi: 10.1038/2271251a0. [DOI] [PubMed] [Google Scholar]
  5. Baur S., Vitetta E. S., Sherr C. J., Schenkein I., Uhr J. W. Isolation of heavy and light chains of immunoglobulin from the surfaces of lymphoid cells. J Immunol. 1971 Apr;106(4):1133–1135. [PubMed] [Google Scholar]
  6. Blyden G., Handschumacher R. E. Purification and properties of human lymphocyte activating factor (LAF). J Immunol. 1977 May;118(5):1631–1638. [PubMed] [Google Scholar]
  7. Boswell H. S., Ahmed A., Scher I., Singer A. Role of accessory cells in B cell activation. II. The interaction of B cells with accessory cells results in the exclusive activation of an Lyb5+ B cell subpopulation. J Immunol. 1980 Sep;125(3):1340–1348. [PubMed] [Google Scholar]
  8. Cambier J. C., Ligler F. S., Uhr J. W., Kettman J. R., Vitetta E. S. Blocking of primary in vitro antibody responses to thymus-independent and thymus-dependent antigens with antiserum specific for IgM or IgD. Proc Natl Acad Sci U S A. 1978 Jan;75(1):432–435. doi: 10.1073/pnas.75.1.432. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cambier J. C., Neale M. J. Isolated phosphorylcholine binding lymphocytes. I. Use of a cleavable crosslinking reagent for solid-phase adsorbent isolation of functional antigen binding cells. J Immunol Methods. 1982 Jun 11;51(2):209–221. doi: 10.1016/0022-1759(82)90260-5. [DOI] [PubMed] [Google Scholar]
  10. Cambier J. C., Vitetta E. S., Uhr J. W., Kettman J. R. B-cell tolerance. II. Trinitrophenyl human gamma globulin-induced tolerance in adult and neonatal murine B cells responsive to thymus-dependent and independent forms of the same hapten. J Exp Med. 1977 Mar 1;145(3):778–783. doi: 10.1084/jem.145.3.778. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Darzynkiewicz Z., Sharpless T., Staiano-Coico L., Melamed M. R. Subcompartments of the G1 phase of cell cycle detected by flow cytometry. Proc Natl Acad Sci U S A. 1980 Nov;77(11):6696–6699. doi: 10.1073/pnas.77.11.6696. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Farrar J. J., Fuller-Farrar J., Simon P. L., Hilfiker M. L., Stadler B. M., Farrar W. L. Thymoma production of T cell growth factor (Interleukin 2). J Immunol. 1980 Dec;125(6):2555–2558. [PubMed] [Google Scholar]
  13. Fidler J. M. The induction of hapten-specific immunological tolerance and immunity in B lymphocytes. VI. Differential tolerance susceptibility in adult spleen as a function of B-cell maturation level. J Exp Med. 1979 Sep 19;150(3):491–506. doi: 10.1084/jem.150.3.491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fuller-Farrar J., Hilfiker M. L., Farrar W. L., Farrar J. J. Phorbol myristic acetate enhances the production of interleukin 2. Cell Immunol. 1981 Feb;58(1):156–164. doi: 10.1016/0008-8749(81)90157-x. [DOI] [PubMed] [Google Scholar]
  15. Haas W., Layton J. E. Separation of antigen-specific lymphocytes. I. Enrichment of antigen-binding cells. J Exp Med. 1975 May 1;141(5):1004–1014. doi: 10.1084/jem.141.5.1004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Haas W. Separation of antigen-specific lymphocytes. II. Enrichment of hapten-specific antibody-forming cell precursors. J Exp Med. 1975 May 1;141(5):1015–1029. doi: 10.1084/jem.141.5.1015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Haskill J. S. Density distribution analysis of antigen-sensitive cells in the rat. J Exp Med. 1969 Oct 1;130(4):877–893. doi: 10.1084/jem.130.4.877. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jennings J. J., Rittenberg M. B. Evidence for separate subpopulations of B cells responding to T-independent and T-dependent immunogens. J Immunol. 1976 Nov;117(5 PT2):1749–1752. [PubMed] [Google Scholar]
  19. Julius M. H., Masuda T., Herzenberg L. A. Demonstration that antigen-binding cells are precursors of antibody-producing cells after purification with a fluorescence-activated cell sorter. Proc Natl Acad Sci U S A. 1972 Jul;69(7):1934–1938. doi: 10.1073/pnas.69.7.1934. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kessler S. W. Rapid isolation of antigens from cells with a staphylococcal protein A-antibody adsorbent: parameters of the interaction of antibody-antigen complexes with protein A. J Immunol. 1975 Dec;115(6):1617–1624. [PubMed] [Google Scholar]
  21. Kettman J., Dutton R. W. An in vitro primary immune response to 2,4,6-trinitrophenyl substituted erythrocytes: response against carrier and hapten. J Immunol. 1970 Jun;104(6):1558–1561. [PubMed] [Google Scholar]
  22. Kiefer H. Separation of antigen-specific lymphocytes. A new general method of releasing cells bound to nylon mesh. Eur J Immunol. 1975 Sep;5(9):624–628. doi: 10.1002/eji.1830050909. [DOI] [PubMed] [Google Scholar]
  23. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  24. Melchers F., Andersson J., Lernhardt W., Schreier M. H. Roles of surface-bound immunoglobulin molecules in regulating the replication and maturation to immunoglobulin secretion of B lymphocytes. Immunol Rev. 1980;52:89–114. doi: 10.1111/j.1600-065x.1980.tb00332.x. [DOI] [PubMed] [Google Scholar]
  25. Mishell R. I., Dutton R. W. Immunization of dissociated spleen cell cultures from normal mice. J Exp Med. 1967 Sep 1;126(3):423–442. doi: 10.1084/jem.126.3.423. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Monroe J. G., Cambier J. C. Cell cycle dependence for expression of membrane associated IgD, IgM and Ia antigen on mitogen-stimulated murine B-lymphocytes. Ann N Y Acad Sci. 1982;399:238–254. doi: 10.1111/j.1749-6632.1982.tb25677.x. [DOI] [PubMed] [Google Scholar]
  27. Monroe J. G., Havran W. L., Cambier J. C. Enrichment of viable lymphocytes in defined cycle phases by sorting on the basis of pulse width of axial light extinction. Cytometry. 1982 Jul;3(1):24–27. doi: 10.1002/cyto.990030107. [DOI] [PubMed] [Google Scholar]
  28. Mosier D. E., Scher I., Paul W. E. In vitro responses of CBA/N mice: spleen cells of mice with an X-linked defect that precludes immune responses to several thymus-independent antigens can respond to TNP-lipopolysaccharide. J Immunol. 1976 Oct;117(4):1363–1369. [PubMed] [Google Scholar]
  29. Parker D. C. Stimulation of mouse lymphocytes by insoluble anti-mouse immunoglobulin. Nature. 1975 Nov 27;258(5533):361–363. doi: 10.1038/258361a0. [DOI] [PubMed] [Google Scholar]
  30. Quintáns J., Cosenza H. Antibody response to phosphorylcholine in vitro. II. Analysis of T-dependent and T-independent responses. Eur J Immunol. 1976 Jun;6(6):399–405. doi: 10.1002/eji.1830060605. [DOI] [PubMed] [Google Scholar]
  31. SELL S., GELL P. G. STUDIES ON RABBIT LYMPHOCYTES IN VITRO. I. STIMULATION OF BLAST TRANSFORMATION WITH AN ANTIALLOTYPE SERUM. J Exp Med. 1965 Aug 1;122:423–440. doi: 10.1084/jem.122.2.423. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Schimpl A., Wecker E., Hünig T. Mechanism of T-cell help in the immune response to soluble protein antigens. I. Evidence for in situ generation and action of T-cell-replacing factor during the anamnestic response to dinitrophenyl keyhole limpet hemocyanin in vitro. J Exp Med. 1977 May 1;145(5):1216–1227. doi: 10.1084/jem.145.5.1216. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Scott D. W., Alexander C. B-cell subsets responsive to fluorescein-conjugated antigens. II. "Cross-priming" and its elimination by bromodeoxyuridine and light. Cell Immunol. 1980 Aug 1;53(2):376–381. doi: 10.1016/0008-8749(80)90337-8. [DOI] [PubMed] [Google Scholar]
  34. Scott D. W., Venkataraman M., Jandinski J. J. Multiple pathways of B lymphocyte tolerance. Immunol Rev. 1979;43:241–280. doi: 10.1111/j.1600-065x.1979.tb00424.x. [DOI] [PubMed] [Google Scholar]
  35. Sell S., Rowe D. S., Gell P. G. Studies on rabbit lymphocytes in vitro. 3. Proteins, RNA, and DNA synthesis by lymphocyte cultures after stimulation with phytohaemagglutinin, with staphylococcal filtrate, with antiallotype serum, and with heterologous antiserum to rabbit whole serum. J Exp Med. 1965 Oct 1;122(4):823–839. doi: 10.1084/jem.122.4.823. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Sieckmann D. G., Scher I., Asofsky R., Mosier D. E., Paul W. E. Activation of mouse lymphocytes by anti-immunoglobulin. II. A thymus-independent response by a mature subset of B lymphocytes. J Exp Med. 1978 Dec 1;148(6):1628–1643. doi: 10.1084/jem.148.6.1628. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Tite J. P., Marshall-Clarke S., Playfair J. H. Differential tolerance of thymus-independent and thymus-dependent antibody responses. Immunology. 1978 Feb;34(2):337–345. [PMC free article] [PubMed] [Google Scholar]
  38. Vaux D. L., Pike B. L., Nossal G. J. Antibody production by single, hapten-specific B lymphocytes: an antigen-driven cloning system free of filler or accessory cells. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7702–7706. doi: 10.1073/pnas.78.12.7702. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Venkataraman M., Scott D. W. Cellular events in tolerance. VIII. Analysis of the proliferative capacity of antigen-binding cells isolated from normal or tolerant spleens. J Immunol. 1980 Feb;124(2):607–610. [PubMed] [Google Scholar]
  40. Wetzel G. D., Kettman J. R. Activation of murine B lymphocytes. III. Stimulation of B lymphocyte clonal growth with lipopolysaccharide and dextran sulfate. J Immunol. 1981 Feb;126(2):723–728. [PubMed] [Google Scholar]
  41. YOUNG C. W., HODAS S. HYDROXYUREA: INHIBITORY EFFECT ON DNA METABOLISM. Science. 1964 Nov 27;146(3648):1172–1174. doi: 10.1126/science.146.3648.1172. [DOI] [PubMed] [Google Scholar]
  42. Zitron I. M., Mosier D. E., Paul W. E. The role of surface IgD in the response to thymic-independent antigens. J Exp Med. 1977 Dec 1;146(6):1707–1718. doi: 10.1084/jem.146.6.1707. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES