Role of BCR affinity in T cell–dependent antibody responses in vivo

Abstract

Antibody affinity for antigen is believed to govern B lymphocyte selection during T-dependent immune responses. To examine antibody affinity in T cell–dependent immune responses, we compared mice that carry targeted VHB1-8 antibody genes with high or low antigen-binding affinity. We found that high- and low-affinity B cells had the same intrinsic capacity to respond to antigen, but in experiments where limiting numbers of high- and low-affinity B cells were mixed in wild-type recipient mice, only the high-affinity B cells accumulated in germinal centers (GCs). In GCs, high-affinity B cells accumulated fewer VH somatic mutations than low affinity B cells. This effect was due to selections as the frequency of mutation in noncoding immunoglobulin gene DNA is the same in high- and low- affinity B cells. Thus, B cells recruited to the GC appeared to undergo a fixed mutation program, regardless of initial B cell receptor affinity. We conclude that in addition to the selection that occurs in GCs, stringent selection for high-affinity clones is also imposed in the early stages of the T cell–dependent immune response in vivo.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: NP-specific antibody responses.
Figure 2: GC response, as measured by flow cytometry.
Figure 3: Immunohistological staining of GCs.
Figure 4: Somatic mutation in B1-8hi and B1-8lo day 25 GC B cells.
Figure 5: Clonal selection during T-dependent immune responses.
Figure 6: Marginal zone B cell development.

References

  1. 1

    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. 176, 679–687 (1992).

  2. 2

    MacLennan, I.C., Liu, Y.J. & Johnson, G.D. Maturation and dispersal of B-cell clones during T cell-dependent antibody responses. Immunol. Rev. 126, 143–161 (1992).

  3. 3

    Jacob, J., Kassir, R. & Kelsoe, G. In situ studies of the primary immune response to (4-hydroxy-3-nitrophenyl)acetyl. I. The architecture and dynamics of responding cell populations. J. Exp. Med. 173, 1165–1175 (1991).

  4. 4

    Liu, Y.J., Zhang, J., Lane, P.J., Chan, E.Y. & MacLennan, I.C. Sites of specific B cell activation in primary and secondary responses to T cell-dependent and T cell-independent antigens. Eur. J. Immunol. 21, 2951–2962 (1991).

  5. 5

    Berek, C., Griffiths, G.M. & Milstein, C. Molecular events during maturation of the immune response to oxazolone. Nature 316, 412–418 (1985).

  6. 6

    Cumano, A. & Rajewsky, K. Clonal recruitment and somatic mutation in the generation of immunological memory to the hapten NP. EMBO J. 5, 2459–2468 (1986).

  7. 7

    Kocks, C. & Rajewsky, K. Stable expression and somatic hypermutation of antibody V regions in B-cell developmental pathways. Annu. Rev. Immunol. 7, 537–559 (1989).

  8. 8

    Clarke, S.H. et al. V region gene usage and somatic mutation in the primary and secondary responses to influenza virus hemagglutinin. J. Immunol. 144, 2795–2801 (1990).

  9. 9

    Griffiths, G.M., Berek, C., Kaartinen, M. & Milstein, C. Somatic mutation and the maturation of immune response to 2-phenyl oxazolone. Nature 312, 271–275 (1984).

  10. 10

    Berek, C. & Milstein, C. Mutation drift and repertoire shift in the maturation of the immune response. Immunol. Rev. 96, 23–41 (1987).

  11. 11

    McKean, D. et al. Generation of antibody diversity in the immune response of BALB/c mice to influenza virus hemagglutinin. Proc. Natl. Acad. Sci. USA 81, 3180–3184 (1984).

  12. 12

    Kocks, C. & Rajewsky, K. Stepwise intraclonal maturation of antibody affinity through somatic hypermutation. Proc. Natl. Acad. Sci. USA 85, 8206–8210 (1988).

  13. 13

    Dal Porto, J.M., Haberman, A.M., Shlomchik, M.J. & Kelsoe, G. Antigen drives very low affinity B cells to become plasmacytes and enter germinal centers. J. Immunol. 161, 5373–5381 (1998).

  14. 14

    Allen, D. et al. Timing, genetic requirements and functional consequences of somatic hypermutation during B-cell development. Immunol. Rev. 96, 5–22 (1987).

  15. 15

    Weiss, U. & Rajewsky, K. The repertoire of somatic antibody mutants accumulating in the memory compartment after primary immunization is restricted through affinity maturation and mirrors that expressed in the secondary response. J. Exp. Med. 172, 1681–1689 (1990).

  16. 16

    Berek, C., Berger, A. & Apel, M. Maturation of the immune response in germinal centers. Cell 67, 1121–1129 (1991).

  17. 17

    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. 7, 1995–2001 (1988).

  18. 18

    Rogozin, I.B. & Kolchanov, N.A. Somatic hypermutagenesis in immunoglobulin genes. II. Influence of neighbouring base sequences on mutagenesis. Biochem. Biophys. Acta 1171, 11–18 (1992).

  19. 19

    Betz, A.G., Rada, C., Pannell, R., Milstein, C. & Neuberger, M.S. Passenger transgenes reveal intrinsic specificity of the antibody hypermutation mechanism: clustering, polarity, and specific hot spots. Proc. Natl. Acad. Sci. USA 90, 2385–2388 (1993).

  20. 20

    Eisen, H.N. & Siskind, G.W. Variations in affinities of antibodies during the immune response. Biochemistry 3, 996–1008 (1964).

  21. 21

    Jerne, N.K. A study of avidity based on rabbit skin responses to diptheria toxin antitoxin mixtures. Acta Pathol. Microbiol. Scand. 87 (Suppl.) 1–183 (1951).

  22. 22

    Siskind, G.W. & Benacerraf, B. Cell selection by antigen in the immune response. Adv. Immunol. 10, 1–50 (1969).

  23. 23

    French, D.L., Laskov, R. & Scharff, M.D. The role of somatic hypermutation in the generation of antibody diversity. Science 244, 1152–1157 (1989).

  24. 24

    Weiss, U., Zoebelein, R. & Rajewsky, K. Accumulation of somatic mutants in the B cell compartment after primary immunization with a T cell-dependent antigen. Eur. J. Immunol. 22, 511–517 (1992).

  25. 25

    Shih, T.-A.Y., Roederer, M. & Nussenzweig, M.C. Role of antigen receptor affinity in T cell-independent antibody responses in vivo. Nature Immunol. 3, 399–406 (2002)

  26. 26

    Nagaoka, H., Gonzalez-Aseguinolaza, G., Tsuji, M. & Nussenzweig, M.C. Immunization and infection change the number of recombination activating gene (RAG)-expressing B cells in the periphery by altering immature lymphocyte production. J. Exp. Med. 191, 2113–2120 (2000).

  27. 27

    Smith, K.G., Nossal, G.J. & Tarlinton, D.M. FAS is highly expressed in the germinal center but is not required for regulation of the B-cell response to antigen. Proc. Natl. Acad. Sci. USA 92, 11628–11632 (1995).

  28. 28

    Watanabe, D., Suda, T. & Nagata, S. Expression of Fas in B cells of the mouse germinal center and Fas-dependent killing of activated B cells. Int. Immunol. 7, 1949–1956 (1995).

  29. 29

    Jacob, J., Kelsoe, G., Rajewsky, K. & Weiss, U. Intraclonal generation of antibody mutants in germinal centres. Nature 354, 389–392 (1991).

  30. 30

    Toellner, K.-M. et al. Low-level Hypermutation in T cell–independent germinal centers compared with high mutation rates associated with T cell-dependent germinal centers. J. Exp. Med. 195, 383–389 (2002).

  31. 31

    MacLennan, I.C. Germinal centers. Annu. Rev. Immunol. 12, 117–139 (1994).

  32. 32

    Rada, C., Ehrenstein, M.R., Neuberger, M.S. & Milstein, C. Hot spot focusing of somatic hypermutation in MSH2-deficient mice suggests two stages of mutational targeting. Immunity 9, 135–141 (1998).

  33. 33

    Jacob, J., Przylepa, J., Miller, C. & Kelsoe, G. In situ studies of the primary immune response to (4-hydroxy-3-nitrophenyl)acetyl. III. The kinetics of V region mutation and selection in germinal center B cells. J. Exp. Med. 178, 1293–307 (1993).

  34. 34

    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. 22, 3001–3011 (1992).

  35. 35

    McHeyzer-Williams, M.G. & Ahmed, R. B cell memory and the long-lived plasma cell. Curr. Opin. Immunol. 11, 172–179 (1999).

  36. 36

    Ziegner, M., Steinhauser, G. & Berek, C. Development of antibody diversity in single germinal centers: selective expansion of high-affinity variants. Eur. J. Immunol. 24, 2393–2400 (1994).

  37. 37

    Liu, Y.J., Oldfield, S. & MacLennan, I.C. Memory B cells in T cell–dependent antibody responses colonize the splenic marginal zones. Eur. J. Immunol. 18, 355–362 (1988).

  38. 38

    Neuberger, M.S. et al. Memory in the B-cell compartment: antibody affinity maturation. Phil. Trans. R. Soc. Lond. B 355, 357–360 (2000).

  39. 39

    Rajewsky, K. Clonal selection and learning in the antibody system. Nature 381, 751–758 (1996).

  40. 40

    Kroese, F.G., Wubbena, A.S., Seijen, H.G. & Nieuwenhuis, P. Germinal centers develop oligoclonally. Eur. J. Immunol. 17, 1069–1072 (1987).

  41. 41

    Bachmann, M.F. et al. The influence of antigen organization on B cell responsiveness. Science 262, 1448–1451 (1993).

  42. 42

    Batista, F.D. & Neuberger, M.S. Affinity dependence of the B cell response to antigen: a threshold, a ceiling, and the importance of off-rate. Immunity 8, 751–759 (1998).

  43. 43

    Dintzis, H.M., Dintzis, R.Z. & Vogelstein, B. Molecular determinants of immunogenicity: the immunon model of immune response. Proc. Natl. Acad. Sci. USA 73, 3671–3675 (1976).

  44. 44

    Fischer, M.B. et al. Dependence of germinal center B cells on expression of CD21/CD35 for survival. Science 280, 582–585 (1998).

  45. 45

    Foote, J. & Eisen, H.N. Kinetic and affinity limits on antibodies produced during immune responses. Proc. Natl. Acad. Sci. USA 92, 1254–1256 (1995).

  46. 46

    Foote, J. & Milstein, C. Kinetic maturation of an immune response. Nature 352, 530–2 (1991).

  47. 47

    Kouskoff, V. et al. Antigens varying in affinity for the B cell receptor induce differential B lymphocyte responses. J. Exp. Med. 188, 1453–1464 (1998).

  48. 48

    Roost, H.P. et al. Early high-affinity neutralizing anti-viral IgG responses without further overall improvements of affinity. Proc. Natl. Acad. Sci. USA 92, 1257–1261 (1995).

  49. 49

    Batista, F.D. & Neuberger, M.S. B cells extract and present immobilized antigen: implications for affinity discrimination. EMBO J. 19, 513–520 (2000).

  50. 50

    Takahashi, Y., Ohta, H. & Takemori, T. Fas is required for clonal selection in germinal centers and the subsequent establishment of the memory B cell repertoire. Immunity 14, 181–192 (2001).

Download references

Acknowledgements

We thank E. Besmer and members of the Nussenzweig lab for helpful comments on the manuscript and Nai-Ying Zheng for histology. We are also grateful to P. Wilson for help with analysis of somatic hypermutation. Supported by NIH MSTP grant GM07739 (to T. Y. S.) and HHMI and grants from the Leukemia Society and NIH (to M. C. N.).

Author information

Correspondence to Michel C. Nussenzweig.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and Permissions

About this article

Further reading