Understanding how memory B cells are induced and relate to long-lived plasma cells is important for vaccine development. Immunity to oral vaccines has been considered short-lived because of a poor ability to develop IgA B-cell memory. Here we demonstrate that long-lived mucosal IgA memory is readily achieved by oral but not systemic immunization in mouse models with NP hapten conjugated with cholera toxin and transfer of B1-8high/GFP+ NP-specific B cells. Unexpectedly, memory B cells are poorly related to long-lived plasma cells and less affinity-matured. They are α4β7-integrin+CD73+PD-L2+CD80+ and at systemic sites mostly IgM+, while 80% are IgA+ in Peyer's patches. On reactivation, most memory B cells in Peyer's patches are GL7−, but expand in germinal centres and acquire higher affinity and more mutations, demonstrating strong clonal selection. CCR9 expression is found only in Peyer's patches and appears critical for gut homing. Thus, gut mucosal memory possesses unique features not seen after systemic immunization.
The germinal center (GC) reaction in Peyer′s patches (PP) requires continuous access to antigens, but how this is achieved is not known. Here we show that activated antigen-specific CCR6 + CCR1 + GL7 − B cells make close contact with M cells in the subepithelial dome (SED). Using in situ photoactivation analysis of antigen-specific SED B cells, we find migration of cells towards the GC. Following antigen injection into ligated intestinal loops containing PPs, 40% of antigen-specific SED B cells bind antigen within 2 h, whereas unspecifc cells do not, indicating B cell-receptor involvment. Antigen-loading is not observed in M cell-deficient mice, but is unperturbed in mice depleted of classical dendritic cells (DC). Thus, we report a M cell-B cell antigen-specific transporting pathway in PP that is independent of DC. We propose that this antigen transporting pathway has a critical role in gut IgA responses, and should be taken into account when developing mucosal vaccines.
Recent debate over the ability to generate memory B cells in the gut immune system prompted us to explore a novel model with transgenic NP-hapten-specific GFP+ B cells and oral immunizations with NP-conjugated to cholera toxin (NP-CT). We found that orally immunized mice developed exceptional long term memory B cell populations that resided in B cell follicles in Peyer’s patches, mesenteric lymph nodes and spleen. A majority of the GFP+ memory cells, defined by CD80, CD73 and PD-L2 expression, were IgM+, but also a significant number were IgA+ and in spleen IgG+ memory B cells were frequent. Upon analysis of the NP binding VH186.2 IgA gene sequences between 20-40 % carried high affinity mutations and VH186.2 IgG (bone marrow) and IgA (intestine and bone marrow) gene sequences were clonally related. A challenge immunization after 1.5 years, either orally or i.p, elicited a strong NP-specific gut lamina propria (LP) IgA and serum IgG response, starting from a calculated frequency of 1 NP-memory / total 10.000 B cells in these mice. Gut LP IgA plasma cells increased after challenge from 0.05% long-lived NP-specific IgA plasma cells to >5% NP-specific IgA plasma cells of all LP IgA plasma cells. The boosted IgA plasma cells carried more mutations and higher affinities (>60%) than long-lived memory B cells, demonstrating unexpectedly effective selection and maturational processes in gut IgA memory responses.
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