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. 1993 Apr 1;90(7):3088–3092. doi: 10.1073/pnas.90.7.3088

Soluble antigen profoundly reduces memory B-cell numbers even when given after challenge immunization.

G J Nossal 1, M Karvelas 1, B Pulendran 1
PMCID: PMC46242  PMID: 8464928

Abstract

The splenic B-cell repertoire of unimmunized C57BL/6 mice can be examined for anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) B cells of relatively high affinity by using a dual strategy. First, limiting numbers of splenocytes are polyclonally activated by Escherichia coli lipopolysaccharide and a mixture of interleukins 2, 4, and 5 in the presence of 3T3 filler cells, thus ensuring that many B-cell clones switch to IgG1 antibody production. Second, an enzyme-linked immunosorbent assay is geared to register only higher-affinity antibody by (i) detecting only bivalent IgG1 antibody and ignoring IgM and (ii) using a lowly substituted NP-conjugated protein as the capture layer. Naive spleens contain very few higher-affinity anti-NP B cells thus defined, but thymus (T)-dependent immunization causes the appearance of approximately 10(5) per spleen within 2 weeks. The development of these clonable anti-NP antibody-forming cell precursors can be virtually eliminated by a single injection of 1 mg of soluble, freshly deaggregated NP2-human serum albumin (HSA). This toleragen works not only if injected prior to challenge immunization, but even if given up to 6 days later. Soluble HSA works partially but not nearly as well as NP2-HSA, suggesting the possibility that the toleragen must act on T and B cells. NP conjugated to irrelevant carriers achieved partial tolerance in only one of four experiments. The studies demonstrate the need for continuing T-cell help throughout the process of memory B-cell generation. They also show that those recently activated T cells involved in this process can be silenced in vivo by soluble toleragen.

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Selected References

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

  1. Allen D., Cumano A., Dildrop R., Kocks C., Rajewsky K., Rajewsky N., Roes J., Sablitzky F., Siekevitz M. Timing, genetic requirements and functional consequences of somatic hypermutation during B-cell development. Immunol Rev. 1987 Apr;96:5–22. doi: 10.1111/j.1600-065x.1987.tb00506.x. [DOI] [PubMed] [Google Scholar]
  2. Allen D., Simon T., Sablitzky F., Rajewsky K., Cumano A. Antibody engineering for the analysis of affinity maturation of an anti-hapten response. EMBO J. 1988 Jul;7(7):1995–2001. doi: 10.1002/j.1460-2075.1988.tb03038.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Berek C., Berger A., Apel M. Maturation of the immune response in germinal centers. Cell. 1991 Dec 20;67(6):1121–1129. doi: 10.1016/0092-8674(91)90289-b. [DOI] [PubMed] [Google Scholar]
  4. Berek C., Milstein C. Mutation drift and repertoire shift in the maturation of the immune response. Immunol Rev. 1987 Apr;96:23–41. doi: 10.1111/j.1600-065x.1987.tb00507.x. [DOI] [PubMed] [Google Scholar]
  5. Cosgrove D., Gray D., Dierich A., Kaufman J., Lemeur M., Benoist C., Mathis D. Mice lacking MHC class II molecules. Cell. 1991 Sep 6;66(5):1051–1066. doi: 10.1016/0092-8674(91)90448-8. [DOI] [PubMed] [Google Scholar]
  6. Cumano A., Rajewsky K. Clonal recruitment and somatic mutation in the generation of immunological memory to the hapten NP. EMBO J. 1986 Oct;5(10):2459–2468. doi: 10.1002/j.1460-2075.1986.tb04522.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cumano A., Rajewsky K. Structure of primary anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) antibodies in normal and idiotypically suppressed C57BL/6 mice. Eur J Immunol. 1985 May;15(5):512–520. doi: 10.1002/eji.1830150517. [DOI] [PubMed] [Google Scholar]
  8. Erikson J., Radic M. Z., Camper S. A., Hardy R. R., Carmack C., Weigert M. Expression of anti-DNA immunoglobulin transgenes in non-autoimmune mice. Nature. 1991 Jan 24;349(6307):331–334. doi: 10.1038/349331a0. [DOI] [PubMed] [Google Scholar]
  9. Good M. F., Boyd A. W., Nossal G. J. Analysis of true anti-hapten cytotoxic clones in limit dilution microcultures after correction for "anti-self" activity: precursor frequencies, Ly-2 and Thy-1 phenotype, specificity, and statistical methods. J Immunol. 1983 May;130(5):2046–2055. [PubMed] [Google Scholar]
  10. Goodnow C. C., Crosbie J., Adelstein S., Lavoie T. B., Smith-Gill S. J., Brink R. A., Pritchard-Briscoe H., Wotherspoon J. S., Loblay R. H., Raphael K. Altered immunoglobulin expression and functional silencing of self-reactive B lymphocytes in transgenic mice. Nature. 1988 Aug 25;334(6184):676–682. doi: 10.1038/334676a0. [DOI] [PubMed] [Google Scholar]
  11. Jacob J., Kelsoe G. In situ studies of the primary immune response to (4-hydroxy-3-nitrophenyl)acetyl. II. A common clonal origin for periarteriolar lymphoid sheath-associated foci and germinal centers. J Exp Med. 1992 Sep 1;176(3):679–687. doi: 10.1084/jem.176.3.679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Jacob J., Kelsoe G., Rajewsky K., Weiss U. Intraclonal generation of antibody mutants in germinal centres. Nature. 1991 Dec 5;354(6352):389–392. doi: 10.1038/354389a0. [DOI] [PubMed] [Google Scholar]
  13. Karvelas M., Nossal G. J. Memory cell generation ablated by soluble protein antigen by means of effects on T- and B-lymphocyte compartments. Proc Natl Acad Sci U S A. 1992 Apr 1;89(7):3150–3154. doi: 10.1073/pnas.89.7.3150. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lalor P. A., Nossal G. J., Sanderson R. D., McHeyzer-Williams M. G. Functional and molecular characterization of single, (4-hydroxy-3-nitrophenyl)acetyl (NP)-specific, IgG1+ B cells from antibody-secreting and memory B cell pathways in the C57BL/6 immune response to NP. Eur J Immunol. 1992 Nov;22(11):3001–3011. doi: 10.1002/eji.1830221136. [DOI] [PubMed] [Google Scholar]
  15. Linton P. J., Lo D., Lai L., Thorbecke G. J., Klinman N. R. Among naive precursor cell subpopulations only progenitors of memory B cells originate germinal centers. Eur J Immunol. 1992 May;22(5):1293–1297. doi: 10.1002/eji.1830220526. [DOI] [PubMed] [Google Scholar]
  16. Linton P. J., Rudie A., Klinman N. R. Tolerance susceptibility of newly generating memory B cells. J Immunol. 1991 Jun 15;146(12):4099–4104. [PubMed] [Google Scholar]
  17. MacLennan I. C., Gray D. Antigen-driven selection of virgin and memory B cells. Immunol Rev. 1986 Jun;91:61–85. doi: 10.1111/j.1600-065x.1986.tb01484.x. [DOI] [PubMed] [Google Scholar]
  18. MacLennan I. C., Liu Y. J., Johnson G. D. Maturation and dispersal of B-cell clones during T cell-dependent antibody responses. Immunol Rev. 1992 Apr;126:143–161. doi: 10.1111/j.1600-065x.1992.tb00635.x. [DOI] [PubMed] [Google Scholar]
  19. McKean D., Huppi K., Bell M., Staudt L., Gerhard W., Weigert M. Generation of antibody diversity in the immune response of BALB/c mice to influenza virus hemagglutinin. Proc Natl Acad Sci U S A. 1984 May;81(10):3180–3184. doi: 10.1073/pnas.81.10.3180. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Mäkelä O., Karjalainen K. Inherited immunoglobulin idiotypes of the mouse. Immunol Rev. 1977;34:119–138. doi: 10.1111/j.1600-065x.1977.tb00370.x. [DOI] [PubMed] [Google Scholar]
  21. Nemazee D., Buerki K. Clonal deletion of autoreactive B lymphocytes in bone marrow chimeras. Proc Natl Acad Sci U S A. 1989 Oct;86(20):8039–8043. doi: 10.1073/pnas.86.20.8039. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Nossal G. J. Cellular mechanisms of immunologic tolerance. Annu Rev Immunol. 1983;1:33–62. doi: 10.1146/annurev.iy.01.040183.000341. [DOI] [PubMed] [Google Scholar]
  23. Nossal G. J., Karvelas M. Soluble antigen abrogates the appearance of anti-protein IgG1-forming cell precursors during primary immunization. Proc Natl Acad Sci U S A. 1990 Feb;87(4):1615–1619. doi: 10.1073/pnas.87.4.1615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Nossal G. J., Pike B. L. Clonal anergy: persistence in tolerant mice of antigen-binding B lymphocytes incapable of responding to antigen or mitogen. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1602–1606. doi: 10.1073/pnas.77.3.1602. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Nossal G. J., Pike B. L. Single cell studies on the antibody-forming potential of fractionated, hapten-specific B lymphocytes. Immunology. 1976 Feb;30(2):189–202. [PMC free article] [PubMed] [Google Scholar]
  26. Nossal G. J., Riedel C. Sudden appearance of anti-protein IgG1-forming cell precursors early during primary immunization. Proc Natl Acad Sci U S A. 1989 Jun;86(12):4679–4683. doi: 10.1073/pnas.86.12.4679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Nossal G. J. The molecular and cellular basis of affinity maturation in the antibody response. Cell. 1992 Jan 10;68(1):1–2. doi: 10.1016/0092-8674(92)90198-l. [DOI] [PubMed] [Google Scholar]
  28. Rajewsky K. The carrier effect and cellular cooperation in the induction of antibodies. Proc R Soc Lond B Biol Sci. 1971 Jan 12;176(1045):385–392. doi: 10.1098/rspb.1971.0002. [DOI] [PubMed] [Google Scholar]
  29. Rosenberg S. A., Grimm E. A., McGrogan M., Doyle M., Kawasaki E., Koths K., Mark D. F. Biological activity of recombinant human interleukin-2 produced in Escherichia coli. Science. 1984 Mar 30;223(4643):1412–1414. doi: 10.1126/science.6367046. [DOI] [PubMed] [Google Scholar]
  30. Shlomchik M. J., Marshak-Rothstein A., Wolfowicz C. B., Rothstein T. L., Weigert M. G. The role of clonal selection and somatic mutation in autoimmunity. 1987 Aug 27-Sep 2Nature. 328(6133):805–811. doi: 10.1038/328805a0. [DOI] [PubMed] [Google Scholar]
  31. Takebe Y., Seiki M., Fujisawa J., Hoy P., Yokota K., Arai K., Yoshida M., Arai N. SR alpha promoter: an efficient and versatile mammalian cDNA expression system composed of the simian virus 40 early promoter and the R-U5 segment of human T-cell leukemia virus type 1 long terminal repeat. Mol Cell Biol. 1988 Jan;8(1):466–472. doi: 10.1128/mcb.8.1.466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Ternynck T., Avrameas S. Murine natural monoclonal autoantibodies: a study of their polyspecificities and their affinities. Immunol Rev. 1986 Dec;94:99–112. doi: 10.1111/j.1600-065x.1986.tb01166.x. [DOI] [PubMed] [Google Scholar]
  33. Weigert M. G., Cesari I. M., Yonkovich S. J., Cohn M. Variability in the lambda light chain sequences of mouse antibody. Nature. 1970 Dec 12;228(5276):1045–1047. doi: 10.1038/2281045a0. [DOI] [PubMed] [Google Scholar]
  34. Yokota T., Coffman R. L., Hagiwara H., Rennick D. M., Takebe Y., Yokota K., Gemmell L., Shrader B., Yang G., Meyerson P. Isolation and characterization of lymphokine cDNA clones encoding mouse and human IgA-enhancing factor and eosinophil colony-stimulating factor activities: relationship to interleukin 5. Proc Natl Acad Sci U S A. 1987 Nov;84(21):7388–7392. doi: 10.1073/pnas.84.21.7388. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. von Boehmer H., Shortman K. The separation of different cell classes from lymphoid organs. IX. A simple and rapid method for removal of damaged cells from lymphoid cell suspensions. J Immunol Methods. 1973 Apr;2(3):293–301. doi: 10.1016/0022-1759(73)90055-0. [DOI] [PubMed] [Google Scholar]

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