Abstract
The mechanisms controlling affinity maturation have been extensively studied over the last 20 years and the central role of T follicular helper cells (Tfh) in this process has now been clearly established. In order to analyze how Tfh impact on affinity maturation several models have been developed. This chapter aims to present three different techniques to evaluate antigen-specific B cell response and affinity maturation using the NP system: Flow cytometric single cell sorting and sequence analysis, ELISA and ELISpot. They have the advantages of being applicable on all types of mice independently of the presence of a transgenic BCR and to give multiple readout of the antigen-specific immune response and affinity maturation. Although first developed more than 20 years ago, these techniques are still considered to be the gold standard for the analysis of affinity maturation in vivo.
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References
Schwickert TA, Victora GD, Fooksman DR, Kamphorst AO, Mugnier MR, Gitlin AD, Dustin ML, Nussenzweig MC (2011) A dynamic T cell-limited checkpoint regulates affinity-dependent B cell entry into the germinal center. J Exp Med 208(6):1243–1252, doi: jem.20102477 [pii] 10.1084/jem.20102477
Victora GD, Schwickert TA, Fooksman DR, Kamphorst AO, Meyer-Hermann M, Dustin ML, Nussenzweig MC (2010) Germinal center dynamics revealed by multiphoton microscopy with a photoactivatable fluorescent reporter. Cell 143(4):592–605. doi:10.1016/j.cell.2010.10.032
Gitlin AD, Shulman Z, Nussenzweig MC (2014) Clonal selection in the germinal centre by regulated proliferation and hypermutation. Nature 509(7502):637–640. doi:10.1038/nature13300
Linterman MA, Pierson W, Lee SK, Kallies A, Kawamoto S, Rayner TF, Srivastava M, Divekar DP, Beaton L, Hogan JJ, Fagarasan S, Liston A, Smith KG, Vinuesa CG (2011) Foxp3+ follicular regulatory T cells control the germinal center response. Nat Med 17(8):975–982. doi:10.1038/nm.2425
Phan TG, Paus D, Chan TD, Turner ML, Nutt SL, Basten A, Brink R (2006) High affinity germinal center B cells are actively selected into the plasma cell compartment. J Exp Med 203(11):2419–2424, doi: jem.20061254 [pii] 10.1084/jem.20061254
Imanishi T, Makela O (1974) Inheritance of antibody specificity. I. Anti-(4-hydroxy-3-nitrophenyl)acetyl of the mouse primary response. J Exp Med 140(6):1498–1510
Bothwell AL, Paskind M, Reth M, Imanishi-Kari T, Rajewsky K, Baltimore D (1981) Heavy chain variable region contribution to the NPb family of antibodies: somatic mutation evident in a gamma 2a variable region. Cell 24(3):625–637
Cumano A, Rajewsky K (1986) Clonal recruitment and somatic mutation in the generation of immunological memory to the hapten NP. EMBO J 5(10):2459–2468
Allen D, Cumano A, Dildrop R, Kocks C, Rajewsky K, Rajewsky N, Roes J, Sablitzky F, Siekevitz M (1987) Timing, genetic requirements and functional consequences of somatic hypermutation during B-cell development. Immunol Rev 96:5–22
Allen D, Simon T, Sablitzky F, Rajewsky K, Cumano A (1988) Antibody engineering for the analysis of affinity maturation of an anti-hapten response. EMBO J 7(7):1995–2001
McHeyzer-Williams MG, Nossal GJ, Lalor PA (1991) Molecular characterization of single memory B cells. Nature 350(6318):502–505. doi:10.1038/350502a0
Lalor PA, Nossal GJ, Sanderson RD, McHeyzer-Williams MG (1992) 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(11):3001–3011. doi:10.1002/eji.1830221136
Herzenberg LA, Black SJ, Tokuhisa T, Herzenberg LA (1980) Memory B cells at successive stages of differentiation. Affinity maturation and the role of IgD receptors. J Exp Med 151(5):1071–1087
Espeli M, Clatworthy MR, Bokers S, Lawlor KE, Cutler AJ, Kontgen F, Lyons PA, Smith KG (2012) Analysis of a wild mouse promoter variant reveals a novel role for FcgammaRIIb in the control of the germinal center and autoimmunity. J Exp Med 209(12):2307–2319, doi: jem.20121752 [pii] 10.1084/jem.20121752
Goenka R, Matthews AH, Zhang B, O'Neill PJ, Scholz JL, Migone TS, Leonard WJ, Stohl W, Hershberg U, Cancro MP (2014) Local BLyS production by T follicular cells mediates retention of high affinity B cells during affinity maturation. J Exp Med 211(1):45–56. doi:10.1084/jem.20130505
Acknowledgements
This work was supported by a Junior Team Leader starting grant (ME) and a PhD Fellowship (JN) from the Laboratory of Excellence in Research on Medication and Innovative Therapeutics (LabEx LERMIT) supported by a grant from ANR (ANR-10-LABX-33) under the program “Investissements d’Avenir” ANR-11-IDEX-0003-01.
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Natt, J., Espéli, M. (2015). Assessing T Follicular Helper Cell Function In Vivo: Antigen-Specific B Cell Response to Hapten and Affinity Maturation. In: Espéli, M., Linterman, M. (eds) T follicular Helper Cells. Methods in Molecular Biology, vol 1291. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2498-1_8
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DOI: https://doi.org/10.1007/978-1-4939-2498-1_8
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