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.

Most of the immunoglobulin A (IgA) in the gut is generated by B cells in the germinal centers of Peyer's patches through a process that requires the presence of CD4+ follicular B helper T(TFH) cells. The nature of these T(FH) cells in Peyer's patches has been elusive. Here, we demonstrate that suppressive Foxp3+CD4+ T cells can differentiate into TFH cells in mouse Peyer's 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 Peyer's patches promote the selective differentiation of distinct helper T cell subsets, such as TFH cells.

RBP-J is a key mediator of Notch signaling that regulates a large spectrum of cell fate determinations. To elucidate the functions of Notch signaling in T cell development, we inactivated RBP-J specifically at two stages of T cell development by crossing RBP-J floxed mice with lck-cre or CD4-cre transgenic mice. The loss of RBP-J at an earlier developmental stage resulted in enhanced generation and accelerated emigration of gammadelta T cells, whereas alphabeta T cell development was arrested at the double-negative 3 stage. The loss of RBP-J at a later stage did not affect the absolute number or the production rate of CD4 or CD8-positive mature T cells but enhanced Th1 cell response and reduced CD4(+) T cell proliferation. Our data demonstrated that Notch/RBP-J signaling regulates gammadelta T cell generation and migration, alphabeta T cell maturation, terminal differentiation of CD4(+) T cells into Th1/Th2 cells, and activation of T cells.

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.

Mammalian inner ear hair cells in cochleas are believed to be incapable of regeneration after birth, which hampers treatment of sensorineural hearing impairment mainly caused by hair cell loss. Sensory epithelia of cochleas are composed of hair cells and supporting cells, both of which originate from common progenitors. Notch/RBP-J signaling is an evolutionally conserved pathway involved in specification of various cell types in developmental stage and even in some of postnatal mammalian organs. The specification of hair cell fate from the progenitors is inhibited by Notch/RBP-J signaling in embryonic inner ears. However, its function in postnatal inner ears is unknown. We showed that inhibition of Notch/RBP-J signaling, by either conditional disruption of the Rbpsuh gene or treatment with a gamma-secretase inhibitor, could give rise to ectopic hair cells in the supporting cell region in organs of Corti from neonatal mouse cochleas where hair cells have not been considered to regenerate after birth. We also showed that down-regulation of Hes5 and up-regulation of Math1 were associated with ectopic hair cell induction. These results suggest that Notch/RBP-J signaling inhibits supporting cells from differentiation into hair cells even in postnatal days, implying that inhibitors of Notch/RBP-J signaling can be used to help regenerating hair cells after birth and thus serve for potential treatment of intractable sensorineural hearing impairment caused by hair cell loss without genetical manipulation.

Epigenetic changes in chromatin structure at the T helper (Th2) locus correlate with interukin-4 (IL-4) and IL-13 expression during Th2 differentiation. By using a transgenic green fluorescence protein (GFP) reporter system, we show that conserved noncoding sequence-2 (CNS-2), located downstream of the Il4 locus, is a constitutively active enhancer in NKT cells as well as in a subset of CD44(hi) memory phenotype CD4+ T cells. CNS-2 enhancer activity and initial IL-4 expression in CD44(hi) CD4+ T cells were abolished in mice with a CD4-specific deletion of the transcriptional mediator of Notch signaling, Rbp-j. Depletion of CNS-2 active CD4+ T cells markedly decreased Th2 differentiation from naive CD4 T cells and antigen-specific IgE production after in vivo priming. These findings indicate that Notch-regulated CNS-2 enhancer controls initial IL-4 expression in NKT and memory phenotype CD4+ T cells and that CNS-2 active CD44(hi) memory phenotype T cells are important in facilitating Th2 differentiation of naive CD4+ T cells in allergic responses.

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Prostaglandin (PG) E(2) is synthesized from arachidonic acid by cyclooxygenase (COX) and acts as a regulator in ovulation and fertilization reactions. We present the temporal and regional expression patterns of mRNAs for the two Gs-coupled PGE receptors, EP2 and EP4, and for COX-1 and COX-2 in mouse periovulatory follicles and oviducts during superovulation. Analysis using reverse transcription polymerase chain reaction revealed that the mouse ovaries express a significant amount of EP4 mRNA in addition to EP2 mRNA during superovulation. In situ hybridization results revealed that the signals for EP4 mRNA were localized mostly to oocytes in the preantral follicles. Three hours after hCG injection, the signals for EP4 and EP2 mRNA were present in both granulosa and cumulus cells. However, 9 h after hCG injection, just before ovulation, the signals for EP4 mRNA were still detectable in both cell types, whereas those for EP2 mRNA were found only in cumulus cells. COX-2 mRNA expression was present in both granulosa and cumulus cells at 3 h but was present only in cumulus cells at 9 h. COX-1 mRNA expression was not found in granulosa cells at 3 h but was found in these cells at 9 h. In the oviduct, the expression of EP4 and COX-1 mRNA was localized to epithelial cells, whereas expression of EP2 mRNA was localized to the smooth muscle layer. The tightly regulated expression of both EP2 and EP4 in the preovulatory follicles may reflect the essential role of PGE(2) in the ovulation process.