Skip to main content
PMC home page
The Journal of Experimental Medicine logoLink to The Journal of Experimental Medicine
. 1975 May 1;141(5):1015–1029. doi: 10.1084/jem.141.5.1015

Separation of antigen-specific lymphocytes. II. Enrichment of hapten- specific antibody-forming cell precursors

PMCID: PMC2189794  PMID: 165257

Abstract

Normal spleen cells were separated in dishes coated with thin layers of DNP-gelatin or NIP-gelatin into binding and nonbinding cells and stimulated in vitro with DNP- and/or NIP-conjugated polymerized flagellin (POL). Hapten-specific unresponsiveness was induced in the binding cell population by melting the gel at 37 degrees C or in unfractionated cells by pretreatment with soluble hapten-gelatin and could be reversed by treatment with collagenase. A specific enrichment of anti-DNP and anti-NIP antibody-forming cell precursors (AFCP) could be demonstrated in the binding cell populations after treatment with collagenase in cultures with or without "feeder" cells. However, the response of small numbers of unfractionated and purified hapten- specific spleen cells was suboptimal even in the presence of mitomycin- treated or irradiated feeder cells. Optimal numbers of anti-DNP (anti- NIP) antibody-forming cells were generated by small numbers of normal or purified spleen cells in the presence of spleen cells depleted of anti-DNP (anti-NIP) AFCP. In this system the response of only 2 times 10-4 purified hapten-specific cells was higher than the response of 10- 6 unfractionated cells. Purified DNP-specific cells responded only to DNP-POL but not to NIP-POL and purified NIP-specific cells responded only to NIP-POL but not to DNP-POL. The degree of enrichment of anti- DNP AFCP decreased with increasing numbers of binding cells. NIP3- gelatin layers bound four to five times less spleen cells than DNP2- gelatin layers and the enrichment of anti-NIP AFCP (about 300-fold) was three times greater than the enrichment of anti-DNP AFCP (about 100- fold). The immunological significance of hapten-gelatin binding cells which apparently failed to respond to antigen is discussed.

Full Text

The Full Text of this article is available as a PDF (984.7 KB).

Selected References

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

  1. Ada G. L., Byrt P. Specific inactivation of antigen-reactive cells with 125I-labelled antigen. Nature. 1969 Jun 28;222(5200):1291–1292. doi: 10.1038/2221291a0. [DOI] [PubMed] [Google Scholar]
  2. Brody T. Identification of two cell populations required for mouse immunocompetence. J Immunol. 1970 Jul;105(1):126–138. [PubMed] [Google Scholar]
  3. Brownstone A., Mitchison N. A., Pitt-Rivers R. Chemical and serological studies with an iodine-containing synthetic immunological determinant 4-hydroxy-3-iodo-5-nitrophenylacetic acid (NIP) and related compounds. Immunology. 1966 May;10(5):465–479. [PMC free article] [PubMed] [Google Scholar]
  4. Cunningham A. J., Szenberg A. Further improvements in the plaque technique for detecting single antibody-forming cells. Immunology. 1968 Apr;14(4):599–600. [PMC free article] [PubMed] [Google Scholar]
  5. Davie J. M., Paul W. E. Receptors on immunocompetent cells. II. Specificity and nature of receptors on dinitrophenylated guinea pig albumin- 125 I-binding lymphocytes of normal guinea pigs. J Exp Med. 1971 Aug 1;134(2):495–516. doi: 10.1084/jem.134.2.495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Elliott B. E., Haskill J. S., Axelrad M. A. Thymus-derived rosettes are not "helper" cells. J Exp Med. 1973 Nov 1;138(5):1133–1143. doi: 10.1084/jem.138.5.1133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Feldmann M. Induction of immunity and tolerance in vitro by hapten protein conjugates. II. Carrier independence of the response to dinitrophenylated polymerized flagellin. Eur J Immunol. 1972 Apr;2(2):130–137. doi: 10.1002/eji.1830020208. [DOI] [PubMed] [Google Scholar]
  8. Feldmann M. Induction of immunity and tolerance to the dinitrophenyl determinant in vitro. Nat New Biol. 1971 May 5;231(18):21–23. doi: 10.1038/newbio231021a0. [DOI] [PubMed] [Google Scholar]
  9. Fuchs S., Mozes E., Maoz A., Sela M. Thymus independence of a collagen-like synthetic polypeptide and of collagen, and the need for thymus and bone marrow-cell cooperation in the immune response to gelatin. J Exp Med. 1974 Jan 1;139(1):148–158. doi: 10.1084/jem.139.1.148. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Golan D. T., Borel Y. Deletion of hapten-binding cells by a highly radioactive 125-I conjugate. J Exp Med. 1972 Aug 1;136(2):305–317. doi: 10.1084/jem.136.2.305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Haas W., Schrader J. W., Szenberg A. A new, simple method for the preparation of lymphocytes bearing specific receptors. Eur J Immunol. 1974 Aug;4(8):565–570. doi: 10.1002/eji.1830040809. [DOI] [PubMed] [Google Scholar]
  13. Hall W. The functions of immune T and B rosette-forming cells. Immunology. 1973 Aug;25(2):158–196. [PMC free article] [PubMed] [Google Scholar]
  14. Halsall M. K., Makinodan T. Analysis of the limiting-dilution assay used for estimating frequencies of immunocompetent units. Cell Immunol. 1974 Mar 30;11(1-3):456–465. doi: 10.1016/0008-8749(74)90043-4. [DOI] [PubMed] [Google Scholar]
  15. Hamaoka T., Katz D. H. Immunological tolerance in bone marrow-derived lymphocytes. II. Effects of allogeneic cell interactions and enzymatic digestion with trypsin or inactivated hapten-specific precursors of antibody-forming cells. J Exp Med. 1974 Jun 1;139(6):1446–1463. doi: 10.1084/jem.139.6.1446. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Henry C., Kimura J., Wofsy L. Cell separation on affinity columns: the isolation of immunospecific precursor cells from unimmunized mice (lactoside hapten-lymphocyte receptors-immunology). Proc Natl Acad Sci U S A. 1972 Jan;69(1):34–36. doi: 10.1073/pnas.69.1.34. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hunter P., Kettman J. R. Mode of action of a supernatant activity from T-cell cultures that nonspecifically stimulates the humoral immune response. Proc Natl Acad Sci U S A. 1974 Feb;71(2):512–516. doi: 10.1073/pnas.71.2.512. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Katz D. H., Hamaoka T., Benacerraf B. Immunological tolerance in bone marrow-derived lymphocytes. 3. Tolerance induction in primed B cells by hapten conjugates of unrelated immunogenic or "nonimmunogenic" carriers. J Exp Med. 1974 Jun 1;139(6):1464–1472. doi: 10.1084/jem.139.6.1464. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Katz D. H., Hamaoka T., Benacerraf B. Immunological tolerance in bone marrow-derived lymphocytes. I. Evidence for an intracellular mechanism of inactivation of hapten-specific precursors of antibody-forming cells. J Exp Med. 1972 Dec 1;136(6):1404–1429. doi: 10.1084/jem.136.6.1404. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kishimoto T., Ishizaka K. Regulation of antibody response in vitro. IV. Heavy chain antigenic determinants on hapten-specific memory cells. J Immunol. 1972 Dec;109(6):1163–1173. [PubMed] [Google Scholar]
  22. Levine B. B., Levytska V. Hapten-specific insoluble immunoabsorbents prepared from hide powder (collagen). J Immunol. 1969 Mar;102(3):647–655. [PubMed] [Google Scholar]
  23. Moroz L. A., Kotoulas A. O. Cell fractionation on affinity columns. In vivo function of hemocyanin-binding lymphocytes from normal mouse spleen. Int Arch Allergy Appl Immunol. 1973;44(5):657–666. [PubMed] [Google Scholar]
  24. Möller G., Michael G. Frequency of antigen-sensitive cells to thymus-independent antigens. Cell Immunol. 1971 Aug;2(4):309–316. doi: 10.1016/0008-8749(71)90065-7. [DOI] [PubMed] [Google Scholar]
  25. Osoba D. Restriction of the capacity to respond to two antigens by single precursors of antibody-producing cells in culture. J Exp Med. 1969 Jan 1;129(1):141–152. doi: 10.1084/jem.129.1.141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Osoba D. Some physical and radiobiological properties of immunologically reactive mouse spleen cells. J Exp Med. 1970 Aug 1;132(2):368–383. doi: 10.1084/jem.132.2.368. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Raff M. C., Feldmann M., De Petris S. Monospecificity of bone marrow-derived lymphocytes. J Exp Med. 1973 Apr 1;137(4):1024–1030. doi: 10.1084/jem.137.4.1024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Rutishauser U., D'Eustachio P., Edelman G. M. Immunological functions of lymphocytes fractionated with antigen-derivatized fibers. Proc Natl Acad Sci U S A. 1973 Dec;70(12):3894–3898. doi: 10.1073/pnas.70.12.3894. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Siskind G. W., Paul W. E., Benacerraf B. Studies on the effect of the carrier molecule on antihapten antibody synthesis. I. Effect of carrier on the nature of the antibody synthesized. J Exp Med. 1966 Apr 1;123(4):673–688. doi: 10.1084/jem.123.4.673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Wigzell H., Mäkelä O. Separation of normal and immune lymphoid cells by antigen-coated coated columns. Antigen-binding characteristics of membrane antibodies as analyzed by hapten-protein antigens. J Exp Med. 1970 Jul 1;132(1):110–126. doi: 10.1084/jem.132.1.110. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES