Re-utilization of germinal centers in multiple PP results in highly synchronized, oligoclonal and affinity matured gut IgA responses

Whereas recent studies have pointed to multi-centered and diverse gut IgA responses to the microbiota, little information is available on IgA responses following oral immunization with T cell-dependent antigens. Here we have addressed how gut IgA responses develop using a novel approach where NP-hapten was conjugated to cholera toxin (CT), which allowed us to follow, at the molecular level, the site of initiation, expansion, differentiation and distribution of a specific IgA B cell response. Clonal relationships and affinity maturation of specific IgA cells at gut inductive and effector sites were investigated. Unexpectedly, we found gut IgA B cell responses to be oligoclonal and dominated by high affinity maturation. Extensive lineage trees of gut NP-specific IgA cells were generated, revealing strong clonal relationships throughout the entire gut mucosal immune system. Thus, clonally related IgA cells were found in Peyer's patches, mesenteric lymph nodes and the small and large intestine, suggesting an effective expansion and selection process. This was achieved through synchronization of multiple PP hosting the same high affinity B cell clones. Adoptive transfer experiments showed that this was possible through re-utilization of already existing germinal centers (GC) in multiple PP by previously activated GC GL7+ B cells. The re-utilization required that oral antigen was given prior to cell transfer.