Background

Long-lived antibody producing cells, also known as plasmablasts or plasma cells (PCs) primarily originate from germinal centers (GCs), microanatomical sites that form within lymphoid organs following infection or immunization. Entry into the GC reaction involves affinity-based competition of B cells, each expressing an antigen-specific B cell receptor (BCR) with a given affinity and specificity towards the antigen. Upon antigen encounter, B cell activation takes place, which is accompanied by extensive clonal expansion through rapid proliferation. Peyer’s patches (PPs) are lymphoid organs located along the small intestine and are the main site where B cells class switch their immunoglobulins to IgA. The subepithelial dome (SED) is a small niche within the PP wherein immune cells, including B cells, interact with gut-derived antigens. During pre-GC events in PPs, B cells bearing high affinity BCRs do not show preferential advantage in colonization of the SED and formation of PCs, indicating that affinity-based competition does not take place at this site (Biram et al., 2019). Nonetheless, only high affinity clones progress into the GC structures and enter the germinal center reaction. Massive proliferation is a major aspect of the GC reaction in peripheral lymph nodes, spleen and PPs (Victora and Nussenzweig, 2012). During the GC response, B cells undergo iterative cycles of migration between the dark zone (DZ), where they proliferate and mutate their antibody-encoding genes and the light zone (LZ), where the high affinity varaiants are selected by T follicular helper (Tfh) cells for preferential expansion and differentiation into plasma cells. B cells in the GC rapidly divide, and the magnitude of this proliferation is proportional to the strength of T cell help (Gitlin, Shulman and Nussenzweig, 2014). Although B cell proliferation can be induced in vitro by LPS and anti-IgM stimulation, analysis of GC B cell proliferation in culture is currently not possible. In particular, analysis of B cell proliferation in specialized immunological niches such as the SED, can be only examined in vivo.

There are various techniques to measure cell proliferation, which are based on the detection of DNA synthesis, cellular metabolism or proliferation-associated proteins. Cellular metabolism markers such as MTT, XTT and WST-1 assays provide an indirect measurement of cell proliferation and can be inaccurate or in some cases toxic to cells (Liu et al., 1997; Huang et al., 2004). The use of proliferation proteins is a more common and widely used technique and includes staining for Ki67, PCNA and MCM-2 proteins (Bologna-Molina et al., 2013; Carreón-Burciaga et al., 2015). DNA synthesis-based techniques including BrdU, EdU, IdU and CIdU rely on the incorporation of these nucleoside analogs into newly formed DNA strands. As for EdU, the nucleoside incorporation is detected by a click reaction that involves a copper-catalyzed azide-alkyne cycloaddition. DNA replication occurs during S-phase and at this stage, nucleosides are being integrated into the newly formed DNA. Cell cycle progression towards G2/M-phase involves an increase in DNA content. The combination of EdU administration with DNA content staining, which discriminates G1, S and G2/M cells, allows the analysis of proliferation in the different cell-cycle stages. Unlike proliferation-associated protein measurement, DNA synthesis-based measurement, which involves injection of nucleosides into mice, can capture the dynamics of proliferation according to the time allowed for the nucleoside to incorporate into the DNA (Ouadah et al., 2019). In addition, combination of more than one analog, followed by analog-specific detection may provide information on cell populations at different cell cycle stages and on the rate of transition between the stages. Such a method was implemented to study how the magnitude of T cell help affects the speed of the cell cycle within the GC response (Gitlin et al., 2014). Therefore, EdU and other analogs are the preferred method for quantification of proliferating cells within the germinal center. Here, we provide details for EdU incorporation measurement by flow cytometry of B cells in different compartments within the gut associated lymphoid organs. This protocol can be easily adapted to analyze cell proliferation in other experimental systems.