Sidonia Fagarasan

Class Switch Recombination and Hypermutation Require Activation-Induced Cytidine Deaminase (AID), a Potential RNA Editing Enzyme

Induced overexpression of AID in CH12F3-2 B lymphoma cells augmented class switching from IgM to IgA without cytokine stimulation. AID deficiency caused a complete defect in class switching and showed a hyper-IgM phenotype with enlarged germinal centers containing strongly activated B cells before or after immunization. AID-/- spleen cells stimulated in vitro with LPS and cytokines failed to undergo class switch recombination although they expressed germline transcripts. Immunization of AID-/- chimera with 4-hydroxy-3-nitrophenylacetyl (NP) chicken gamma-globulin induced neither accumulation of mutations in the NP-specific variable region gene nor class switching. These results suggest that AID may be involved in regulation or catalysis of the DNA modification step of both class switching and somatic hypermutation.

Foxp3+ follicular regulatory T cells control the germinal center response

Follicular helper (TFH) cells provide crucial signals to germinal center B cells undergoing somatic hypermutation and selection that results in affinity maturation. Tight control of TFH numbers maintains self tolerance. We describe a population of Foxp3+Blimp-1+CD4+ T cells constituting 10–25% of the CXCR5highPD-1highCD4+ T cells found in the germinal center after immunization with protein antigens. These follicular regulatory T (TFR) cells share phenotypic characteristics with TFH and conventional Foxp3+ regulatory T (Treg) cells yet are distinct from both. Similar to TFH cells, TFR cell development depends on Bcl-6, SLAM-associated protein (SAP), CD28 and B cells; however, TFR cells originate from thymic-derived Foxp3+ precursors, not naive or TFH cells. TFR cells are suppressive in vitro and limit TFH cell and germinal center B cell numbers in vivo. In the absence of TFR cells, an outgrowth of non–antigen-specific B cells in germinal centers leads to fewer antigen-specific cells. Thus, the TFH differentiation pathway is co-opted by Treg cells to control the germinal center response.

Aberrant expansion of segmented filamentous bacteria in IgA-deficient gut

The mechanism to maintain homeostasis of the gut microbiota remains largely unknown despite its critical role in the body defense. In the intestines of mice with deficiency of activation-induced cytidine deaminase (AID), the absence of hypermutated IgA is partially compensated for by the presence of large amounts of unmutated IgM and normal expression levels of defensins and angiogenins. We show here a predominant and persistent expansion of segmented filamentous bacteria throughout the small intestine of AID–/– mice. Reconstitution of lamina propria IgA production in AID–/– mice recovered the normal composition of gut flora and abolished the local and systemic activation of the immune system. The results indicate that secretions of IgAs rather than innate defense peptides are critical to regulation of commensal bacterial flora and that the segmented filamentous bacteria antigens are strong stimuli of the mucosal immune system.

Preferential Generation of Follicular B Helper T Cells from Foxp3+ T Cells in Gut Peyer's Patches

Most of the immunoglobulin A (IgA) in the gut is generated by B cells in the germinal centers of Peyers patches through a process that requires the presence of CD4+ follicular B helper T(TFH) cells. The nature of these TFH cells in Peyers patches has been elusive. Here, we demonstrate that suppressive Foxp3+CD4+ T cells can differentiate into TFH cells in mouse Peyers patches. The conversion of Foxp3+ T cells into TFH cells requires the loss of Foxp3 expression and subsequent interaction with B cells. Thus, environmental cues present in gut Peyers patches promote the selective differentiation of distinct helper T cell subsets, such as TFH cells.

Intestinal IgA synthesis: regulation of front-line body defences.

Immunoglobulin A is the most abundant immunoglobulin isotype in mucosal secretions. In this review, we summarize recent advances in our understanding of the sites, mechanisms and functions of intestinal IgA synthesis in mice. On the basis of these recent findings, we propose an updated model for the induction and regulation of IgA responses in the gut. In addition, we discuss new insights into the role of IgA in the maintenance of gut homeostasis and into the reciprocal interactions between gut B cells and the bacterial flora.

In situ class switching and differentiation to IgA-producing cells in the gut lamina propria

One of the front lines of the immune defence is the gut mucosa, where immunoglobulin-α (IgA) is continuously produced to react with commensal bacteria and dietary antigens. It is generally accepted that, after antigenic stimulation in the Peyers patches, IgA+ lymphoblasts (B220+IgA+) migrate through the lymph and blood circulation, and eventually home to the lamina propria of the intestine. Mice that lack activation-induced cytidine deaminase (AID) are defective in class switch recombination (CSR) and somatic hypermutation. CSR changes the immunoglobulin heavy chain constant region (CH) gene being expressed from Cµ to other CH genes, resulting in a switch of the immunoglobulin isotype from IgM to IgG, IgE or IgA. AID-/- mice also secrete large amounts of immunoglobulin-µ (IgM) into faeces, and accumulate B220-IgM+ plasma cells as well as B220+IgM+ cells in the gut. Here we show that lamina propria B220+IgA+ cells have just completed CSR, as they still express both AID and transcripts from circular DNA that has been ‘looped-out’ during CSR. Lamina propria IgM+ B cells seem to be pre-committed to switching to IgA+ in vitro as well as in vivo. Culturing lamina propria IgM+ B cells together with lamina propria stromal cells enhances preferential switching and differentiation of B cells to IgA+ plasma cells. We conclude that IgA+ cells in the gut lamina propria are generated in situ from B220+IgM+ lymphocytes.

Requirement for Lymphoid Tissue-Inducer Cells in Isolated Follicle Formation and T Cell-Independent Immunoglobulin A Generation in the Gut

Immunoglobulin A (IgA) is generated in the gut by both T cell-dependent and T cell-independent processes. The sites and the mechanisms for T cell-independent IgA synthesis remain elusive. Here we show that isolated lymphoid follicles (ILFs) were sites where induction of activation-induced cytidine deaminase (AID) and IgA class switching of B cells took place in the absence of T cells. We also show that formation of ILFs was regulated by interactions between lymphoid tissue-inducer cells expressing the nuclear receptor ROR gamma t (ROR gamma t(+)LTi cells) and stromal cells (SCs). Activation of SCs by ROR gamma t(+)LTi cells through lymphotoxin (LT)-beta receptor (LT beta R) and simultaneously by bacteria through TLRs induced recruitment of dendritic cells (DCs) and B cells and formation of ILFs. These findings provide insight into the crosstalk between bacteria, ROR gamma t(+)LTi cells, SCs, DCs, and B cells required for ILF formation and establish a critical role of ILFs in T cell-independent IgA synthesis in gut.

Adaptive immune regulation in the gut: T cell-dependent and T cell-independent IgA synthesis.

In mammals, the gastrointestinal tract harbors an extraordinarily dense and complex community of microorganisms. The gut microbiota provide strong selective pressure to the host to evolve adaptive immune responses required for the maintenance of local and systemic homeostasis. The continuous antigenic presence in the gut imposes a dynamic remodeling of gut-associated lymphoid tissues (GALT) and the selection of multiple layered strategies for immunoglobulin (Ig) A production. The composite and dynamic gut environment also necessitates heterogeneous, versatile, and convertible T cells, capable of inhibiting (Foxp3(+) T cells) or helping (T(FH) cells) local immune responses. In this review, we describe recent advances in our understanding of dynamic pathways that lead to IgA synthesis, in gut follicular structures and in extrafollicular sites, by T cell-dependent and T cell-independent mechanisms. We discuss the finely tuned regulatory mechanisms for IgA production and emphasize the role of mucosal IgA in the selection and maintenance of the appropriate microbial composition that is necessary for immune homeostasis.

The Inhibitory Receptor PD-1 Regulates IgA Selection and Bacterial Composition in the Gut

Gut Reaction The gut needs to keep its trillions of microbial inhabitants contained. The immune system has evolved a multifaceted approach to this problem, which includes the production of large quantities of immunoglobulin A (IgA) in the intestinal mucosa. In a process that is not well understood, plasma cells that produce IgA specific for the gut microflora are selected in Peyers patches in the gut. Kawamoto et al. (p. 485) used genetically manipulated mice to show that the inhibitory co-receptor, programmed cell death-1 (PD-1), is required for the proper selection IgA-secreting cells in the gut. The effect of PD-1 deletion, however, was not intrinsic to the B cells that produce IgA. Instead, the absence of PD-1 affected the differentiation of T follicular helper cells, which provide important signals to B cells that help guide them as they develop the capacity to produce microflora-specific IgA. Mice deficient in PD-1 exhibited alterations in the composition in their microflora, which suggests that defective selection of IgA can perturb the careful balance that exists between the immune system and resident bacteria. An inhibitory receptor is important for gut microflora containment by immunoglobulin A. Immunoglobulin A (IgA) is essential to maintain the symbiotic balance between gut bacterial communities and the host immune system. Here we provide evidence that the inhibitory co-receptor programmed cell death–1 (PD-1) regulates the gut microbiota through appropriate selection of IgA plasma cell repertoires. PD-1 deficiency generates an excess number of T follicular helper (TFH) cells with altered phenotypes, which results in dysregulated selection of IgA precursor cells in the germinal center of Peyer’s patches. Consequently, the IgAs produced in PD-1–deficient mice have reduced bacteria-binding capacity, which causes alterations of microbial communities in the gut. Thus, PD-1 plays a critical role in regulation of antibody diversification required for the maintenance of intact mucosal barrier.

Regulation of B1 cell migration by signals through Toll-like receptors

Peritoneal B1 cells are known to generate large amounts of antibodies outside their residential site. These antibodies play an important role in the early defense against bacteria and viruses, before the establishment of adaptive immune responses. Although many stimuli, including antigen, lipopolysaccharide, or cytokines, have been shown to activate B1 cells and induce their differentiation into plasma cells, the molecular signals required for their egress from the peritoneal cavity are not understood. We demonstrate here that direct signals through Toll-like receptors (TLRs) induce specific, rapid, and transient down-regulation of integrins and CD9 on B1 cells, which is required for detachment from local matrix and a high velocity movement of cells in response to chemokines. Thus, we revealed an unexpected role for TLRs in governing the interplay between integrins, tetraspanins, and chemokine receptors required for B1 cell egress and, as such, in facilitating appropriate transition from innate to adaptive immune responses.