Unique Maturation Program of the IgE Response In Vivo (Erazo et al. 2007)

In mice, both IgE and IgG1 antibodies are generated during T cell-dependent B cell responses mediated by T helper 2 (Th2) lymphocytes.

T/B monoclonal mice carry anti-chicken ovalbumin (OVA) T cell receptor transgenes (DO11.10) and anti-influenza hemagglutinin (HA) knockin B cell receptor genes on a RAG1-deficient background. Upon immunization with OVA-HA, GC cells were barely detectable in spleen and mesenteric LN 6 days after immunization, but increased rapidly thereafter. The localization of IgG1+ and IgE+ cells in sections of mesenteric LN and spleen was determined by immunohistochemistry. IgG1+ cells occupied the GC areas, and some were also found in the T cell areas and red pulp in the spleen and in the medullary region of LN.

In wild type BALC/c mice, most IgE+ cells were found outside GC areas, in non-GC IgD+ B cell areas, including the medullary region, whereas IgG1+ cells were found mainly in GC.

The highest expression of ɛ sterile transcripts was found in the two GC (PNA+) fractions, indicating that switching to IgE initiates in the GC.

Most IgE+ cells did not express B220 and expressed high amounts of Fas, and a large fraction (80.9% ± 6.3% in T/B monoclonal mice and 57.2% ± 11.5% in Nippostrongylus-infected BALB/c mice) expressed Syndecan-1. Most IgG1+ cells were B220+Fas+Syndecan-1− (96.8% ± 0.9% in T/B monoclonal mice and 94.5% ± 0.5% in Nippostrongylus-infected BALB/c mice), consistent with their GC localization. IgE+Fas+ cells from spleen did not express Pax5, Bcl6, or AID, but expressed high amounts of Blimp and Xbp1. Similar results were obtained with purified IgE+ and IgG1+ cells from lymph nodes.

IgG1+IgE+ dual-expressing cells in similar numbers as IgE+ single-expressing cells. IgG1+IgE+ dual-expressing cells displayed characteristics of plasma cells, very similar to IgE+ single-expressing cells.

Antibody titers of both PEP1-specific IgG1 and IgE increased with time, with the plateau in IgE titers lagging behind the plateau of IgG1 titers. IgG1 and IgE sequences in OVA-PEP1-immunized mice showed increased frequency of the amino acid replacement at positions R97, N100a, and A101 that correlate with high-affinity binding to PEP1. IgG3 antibodies carried a very low number of mutations under these experimental conditions.

A PEP1-specific IgE response could be detected only in sera from mice that received the IgG1+ population. memory IgG1+ B cells are able to generate a high-affinity IgE response. IgG1+ cells and IgD+ cells were purified from T/B monoclonal mice 10 days after immunization with OVA-HA. Most IgG1+ at day 10 are GL7+Fas+B220+ GC cells. The cells were stimulated in vitro with CD40 antibodies, IL-4, and IL-21. The IL-4 concentration used (100 U/ml) is suboptimal to induce CSR to IgE by naive cells but was effective on IgG1+ cells. IgG1+ cell cultures spontaneously secreted IgG1 antibodies but not IgE antibodies. Switching of IgG1+ cells to IgE was profoundly suppressed by IL-21, whereas IgG1 production was not affected.

CD40-CD40L signaling and the transcription factor Bcl6 are essential for the development of GC, SHM, affinity maturation, and B cell memory. CSR to IgE occurs in a GC microenvironment with high IL-4 and low IL-21, perhaps in IgG1+ cells that are exiting GC.

Note: this means anti-CD40, anti-IL-21 and IL-4 culture is effective?

Early activation and CSR occur often in extrafollicular foci, producing short-lived plasma cells that secrete low-affinity antibodies and generate no memory. A second phase occurs in GCs, where CSR, SHM, and affinity maturation take place. GC reactions generate long-lived plasma cells and memory B cells with high-affinity mutated BCR. Memory B cells differentiate into plasma cells upon new antigenic encounter, producing high-affinity antibodies. IgE+ cells do not conform to either the pre-GC (primary foci) or the GC maturation pattern.