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Somatic Hypermutation and Affinity Maturation Analysis Using the 4-Hydroxy-3-Nitrophenyl-Acetyl (NP) System.

Nicole Heise1, Ulf Klein2,3

  • 1Herbert Irving Comprehensive Cancer Center (adjunct), Columbia University (adjunct), 1130 St. Nicholas Avenue R312, New York, NY, 10032, USA.

Methods in Molecular Biology (Clifton, N.J.)
|June 8, 2017
PubMed
Summary

This study establishes a mouse model to measure somatic hypermutation and affinity maturation in the germinal center (GC) reaction. Researchers can now quantify antibody responses and genetic changes during T cell-dependent immunity.

Keywords:
Affinity maturationB cellGerminal centerIg variable region genePlasma cellSomatic hypermutationT cell-dependentT cell-independent

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Area of Science:

  • Immunology
  • Molecular Biology

Background:

  • The germinal center (GC) reaction is crucial for T cell-dependent antibody responses, involving somatic hypermutation and affinity maturation.
  • Measuring these processes requires a well-defined experimental system.

Purpose of the Study:

  • To describe methods for quantifying somatic hypermutation and affinity maturation in the GC response.
  • To establish a system for analyzing antibody affinity maturation using a specific mouse model.

Main Methods:

  • Immunization of C57/Bl6 mice with a hapten (4-hydroxy-3-nitrophenyl-acetyl, NP) coupled to a carrier protein.
  • Quantification of anti-NP plasma cell response using ELISA and ELISPOT analysis.
  • Amplification and sequencing of IgV heavy chain gene segment V186.2 from GC B cells and plasma cells.

Main Results:

  • Identified predominant usage of the V186.2 gene segment in anti-NP B cell responses.
  • Characterized a specific mutation in V186.2 that enhances antibody affinity by approximately 10-fold.
  • Demonstrated the utility of NP-based immunization for measuring plasma cell generation and somatic hypermutation.

Conclusions:

  • The described approaches enable precise measurement of somatic hypermutation and affinity maturation.
  • This system provides a valuable tool for studying the manipulation of GC responses and antibody development.
  • The findings contribute to understanding the molecular mechanisms underlying adaptive immunity.