SpringerProtocols: Abstract: Analysis of the Germinal Center Reaction and In Vivo Long-Lived Plasma Cells

Analysis of the Germinal Center Reaction and In Vivo Long-Lived Plasma Cells

By: Kai-Michael Toellner³, Mahmood Khan³, Daniel Man-Yuen Sze⁴

Abstract

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This chapter describes the analysis of long- and short-lived plasma cells on tissue sections. Mice are immunized with 4-hydroxy-3-nitrophenyl acetyl (NP) coupled to a T-cell-dependent carrier. Antigen-specific germinal center cells and extrafollicular plasma cells are identified by immunohistology on tissue sections. Plasma cells labeled with 5-bromo-2′-deoxyuridine (BrdU) pulses given during different phases of B-cell response are identified on spleen sections. To identify mutated cells originating from cells in germinal centers, antigen-specific cells from spleen sections are isolated by microdissection. From these NP-specific recombined VDJ genes are amplified with family-specific primers by polymerase chain reaction (PCR) for deoxyribonucleic acid (DNA) sequencing. The methods described can be used to characterize origins and lifespan of plasma cells in vivo.

Affiliation(s): (3) MRC Centre for Immune Regulation, Medical School, University of Birmingham, Birmingham, UK
(4) Institute of Haematology, Royal Prince Alfred Hospital, Camperdon, Australia

Book Title: B Cell Protocols

Series: Methods in Molecular Biology | Volume: 271 | Pub. Date: May-12-2004 | Page Range: 111-125 | DOI: 10.1385/1-59259-796-3:111

Subject: Cell Biology

Key Words: Single-cell PCR - DNA sequencing - VDJ amplification - 4-hydroxy-3-nitrophenyl acetyl (NP)-chicken γ-globulin (CGG) - immunoglobulin V gene mutation - carrier priming - BrdU labeling - antigen-specific B cells - immunohistology

References

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1.	Jacob, J., Kassir, R., and Kelsoe, G. (1991) 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.

2.	Liu, Y. J., Zhang, J., Lane, P. J. L., Chan, E. Y. T., and MacLennan, I. C. M. (1991) 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.

3.	Toellner, K.-M., Gulbranson-Judge, A., Taylor, D. R., Sze, D. M.-Y., and MacLennan, I. C. M. (1996) Immunoglobulin switch transcript production in vivo related to the site and time of antigen-specific B cell activation. J. Exp. Med. 183, 2303–2312.

4.	MacLennan, I. C. M., Gulbranson-Judge, A., Toellner, K.-M., et al. (1997) The changing preference of T and B cells for partners as T-dependent antibody responses develop. Immunol. Rev. 156, 53–66.

5.	Ho, F., Lortan, J. E., MacLennan, I. C., and Khan, M. (1986) Distinct short-lived and long-lived antibody-producing cell populations. Eur. J. Immunol. 16, 1297–1301.

6.	Smith, K. G., Hewitson, T. D., Nossal, G. J., and Tarlinton, D. M. (1996) The phenotype and fate of the antibody-forming cells of the splenic foci. Eur. J. Immunol. 26, 444–448.

7.	Sze, D. M.-Y., Toellner, K.-M., Garc’a de Vinuesa, C., Taylor, D. R., and MacLennan, I. C. M. (2000) Intrinsic constraint on plasmablast growth and extrinsic limits of plasma cell survival. J. Exp. Med. 192, 813–821.

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

9.	Stein, H., Bonk, A., Tolksdorf, G., Lennert, K., Rodt, H., and Gerdes, J. (1980) Immunohistologic analysis of the organization of normal lymphoid tissue and non-Hodgkin’s lymphomas. J. Histochem. Cytochem. 28, 746–760.

10.	Jack, R. S., Imanishi-Kari, T., and Rajewsky, K. (1977) Idiotypic analysis of the response of C57BL/6 mice to the (4-hydroxy-3-nitrophenyl)acetyl group. Eur. J. Immunol. 7, 559–565.

11.	Reth, M., Hämmerling, G. J., and Rajewsky, K. ((1978) Analysis of the repertoire of anti-NP antibodies in C57BL/6 mice by cell fusion, I: characterization of antibody families in the primary and hyperimmune response. Eur. J. Immunol. 8, 393–400.

12.	Bothwell, A. L., Paskind, M., Reth, M., Imanishi-Kari, T., Rajewsky, K., and Baltimore, D. (1981) Heavy chain variable region contribution to the NPβ family of antibodies: somatic mutation evident in a λ2a variable region. Cell 24, 625–637 <Occurrence Type="DOI"><Handle>10.1016/0092-8674(81)90089-1</Handle></Occurrence>

13.	McHeyzer-Williams, M. G., Nossal, G. J. V., and Lalor, P. A. (1991) Molecular characterization of single memory B cells. Nature 350, 502–505.

14.	Tao, W., Hardardottir, F., and Bothwell, A. L. (1993) Extensive somatic mutation in the Ig heavy chain V genes in a late primary anti-hapten immune response. Mol. Immunol. 30, 593–602. <Occurrence Type="DOI"><Handle>10.1016/0161-5890(93)90034-9</Handle></Occurrence>

15.	Maizels, N., Lau, J. C., Blier, P. R., and Bothwell, A. (1988) The T cell independent antigen, NP-Ficoll, primes for a high affinity IgM anti-NP response. Mol. Immunol. 25, 1277–1282. <Occurrence Type="DOI"><Handle>10.1016/0161-5890(88)90042-9</Handle></Occurrence>

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

17.	Jacob, J. and Kelsoe, G. (1992) 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.

18.	Manz, R. A., Thiel, A., and Radbruch, A. (1997) Lifetime of plasma cells in the bone marrow. Nature 388, 133–134.

19.	Wynford-Thomas, D. and Williams, E. D. (1986) Use of bromodeoxyuridine for cell kinetic studies in intact animals. Cell Tissue Kinet. 19, 179–182.

20.	Chase, M. W. (1967) Preparation of immunogens. In Williams, C. A. and Chase, M. W., eds., Methods in Immunology and Immunochemistry vol. 1, Academic, New York, pp. 197–209.

21.	Nossal, G. J. and Karvelas, M. (1990) Soluble antigen abrogates the appearance of anti-protein IgG1-forming cell precursors during primary immunization. Proc. Natl. Acad. Sci. USA 87, 1615–1619.

22.	Johnson, G. D. (1989) Immunofluorescence. In Catty, D., ed., Antibodies, A Practical Approach, vol. 2, IRL Press, Oxford, UK, pp. 179–200.

23.	Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

24.	Morris, S. M. (1991) The genetic toxicology of 5-bromodeoxyuridine in mammalian cells. Mutat. Res. 258, 161–188.

25.	Cline, J., Braman, J. C., and Hogrefe, H. H. (1996) PCR fidelity of pfu DNA polymerase and other thermostable DNA polymerases. Nucleic Acids Res. 24, 3546–3551.

26.	Toellner, K.-M., Jenkinson, W. E., Taylor, D. R., Sze, D. M.-Y., Vinuesa, C. G., and MacLennan, I. C. M. (2002) 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.

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