Differentiation and specialization of epithelial cells in the small intestine are regulated in two ways. First, there is differentiation along the crypt-villus axis of the intestinal stem cells into absorptive enterocytes, Paneth, goblet, tuft, enteroendocrine, or M cells, which is mainly regulated by WNT. Second, there is specialization along the cephalocaudal axis with different absorptive and digestive functions in duodenum, jejunum, and ileum that is controlled by several transcription factors such as GATA4. However, so far it is unknown whether location-specific functional properties are intrinsically programmed within stem cells or if continuous signaling from mesenchymal cells is necessary to maintain the location-specific identity of the small intestine. Using the pure epithelial organoid technique, we show that region-specific gene expression profiles are conserved throughout long-term cultures of both mouse and human intestinal stem cells and correlated with differential Gata4 expression. Furthermore, the human organoid culture system demonstrates that Gata4-regulated gene expression is only allowed in absence of WNT signaling. These data show that location-specific function is intrinsically programmed in the adult stem cells of the small intestine and that their differentiation fate is independent of location-specific extracellular signals. In light of the potential future clinical application of small intestine-derived organoids, our data imply that it is important to generate GATA4-positive and GATA4-negative cultures to regenerate all essential functions of the small intestine. Stem Cells 2014;32:1083–1091

Peyer's patches consist of domains of specialized intestinal epithelium overlying gut-associated lymphoid tissue (GALT). Luminal antigens reach the GALT by translocation through epithelial gatekeeper cells, the so-called M cells. We recently demonstrated that all epithelial cells required for the digestive functions of the intestine are generated from Lgr5-expressing stem cells. Here, we show that M cells also derive from these crypt-based Lgr5 stem cells. The Ets family transcription factor SpiB, known to control effector functions of bone marrow-derived immune cells, is specifically expressed in M cells. In SpiB^−/− mice, M cells are entirely absent, which occurs in a cell-autonomous fashion. It has been shown that Tnfsf11 (RankL) can induce M cell development in vivo. We show that in intestinal organoid (“minigut”) cultures, stimulation with RankL induces SpiB expression within 24 h and expression of other M cell markers subsequently. We conclude that RankL-induced expression of SpiB is essential for Lgr5 stem cell-derived epithelial precursors to develop into M cells.

Integrin-mediated adhesion and B cell antigen receptor (BCR) signaling play a critical role in B cell development and function, including antigen-specific B cell differentiation. Here we show that the BCR controls integrin α4β1 (VLA-4)-mediated adhesion of B cells to vascular cell adhesion molecule-1 and fibronectin. Molecular dissection of the underlying signaling mechanism by a combined biochemical, pharmacological, and genetic approach demonstrates that this BCR-controlled integrin-mediated adhesion requires the (consecutive) activation of Lyn, Syk, phosphatidylinositol 3-kinase, Bruton9s tyrosine kinase (Btk), phospholipase C (PLC)γ2, IP3R-mediated Ca²⁺ release, and PKC. In contrast, activation of mitogen-activated protein kinase kinase (MEK) or extracellular signal–regulated kinase (ERK) is not required, and simultaneous activation of MEK, ERK, and PKB is not sufficient either. Furthermore, Btk is also involved in the control of integrin-mediated adhesion of preB cells. The control of integrin α4β1-mediated B cell adhesion by the BCR involves cytoskeletal reorganization and integrin clustering. These results reveal a novel function for the BCR and Btk, i.e., regulation of integrin α4β1 activity, thereby providing new insights into the control of B cell development and differentiation, as well as into the pathogenesis of the immunodeficiency disease X-linked agammaglobulineamia (XLA).

CD4⁺Foxp3⁺ regulatory T cells (T_reg cells) are largely autoreactive yet escape clonal deletion in the thymus. We demonstrate here that CD27–CD70 co-stimulation in the thymus rescues developing T_reg cells from apoptosis and thereby promotes T_reg cell generation. Genetic ablation of CD27 or its ligand CD70 reduced T_reg cell numbers in the thymus and peripheral lymphoid organs, whereas it did not alter conventional CD4⁺Foxp3⁻ T cell numbers. The CD27–CD70 pathway was not required for pre-T_reg cell generation, Foxp3 induction, or mature T_reg cell function. Rather, CD27 signaling enhanced positive selection of T_reg cells within the thymus in a cell-intrinsic manner. CD27 signals promoted the survival of thymic T_reg cells by inhibiting the mitochondrial apoptosis pathway. CD70 was expressed on Aire⁻ and Aire⁺ medullary thymic epithelial cells (mTECs) and on dendritic cells (DCs) in the thymic medulla. CD70 on both mTECs and DCs contributed to T_reg cell development as shown in BM chimera experiments with CD70-deficient mice. In vitro experiments indicated that CD70 on the CD8α⁺ subset of thymic DCs promoted T_reg cell development. Our data suggest that mTECs and DCs form dedicated niches in the thymic medulla, in which CD27–CD70 co-stimulation rescues developing T_reg cells from apoptosis, subsequent to Foxp3 induction by TCR and CD28 signals.