Abstract
Somatic hypermutation of immunoglobulin variable region (IgV) genes and affinity maturation of the antibody response are the hallmarks of the germinal center (GC) reaction in T cell-dependent immune responses. Determining the consequences of the experimental manipulation of the GC response on somatic hypermutation and affinity maturation requires the availability of a system that allows measuring these parameters. Immunization of mice of the C57/Bl6 genetic background with the hapten 4-hydroxy-3-nitrophenyl-acetyl (NP) coupled to a carrier protein leads to the predominant usage of one particular IgV heavy chain gene segment, V186.2, among the responding B cells. Moreover, a specific somatic mutation in codon 33 of V186.2 that leads to a tryptophan to leucine amino acid exchange increases the affinity of the corresponding antibody by ~10-fold, thus representing a molecular marker for affinity maturation. In addition, due to the simplicity of the antigen and the virtual absence of NP-specific plasma cells prior to immunization, NP-based immunizations represent ideal tools to quantify the plasma cell response by measuring NP-specific antisera by ELISA and the generation of NP-specific plasma cells by ELISPOT analysis. We here describe approaches to (1) measure the anti-NP plasma cell response by ELISA and ELISPOT analysis, and to (2) amplify and sequence V186.2 rearrangements from GC B cells and plasma cells to determine the level of somatic hypermutation and the extent of affinity maturation in the anti-NP response.
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Acknowledgments
We thank Nilushi De Silva, Kathryn Silva, and Qiong Shen for their involvement in the establishment of the protocols described here. This work was supported by the NCI/NIH and a fellowship of the German Research Council (DFG) to NH.
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Heise, N., Klein, U. (2017). Somatic Hypermutation and Affinity Maturation Analysis Using the 4-Hydroxy-3-Nitrophenyl-Acetyl (NP) System. In: Calado, D. (eds) Germinal Centers. Methods in Molecular Biology, vol 1623. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7095-7_16
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DOI: https://doi.org/10.1007/978-1-4939-7095-7_16
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