Yoshifumi Yokota

Development of peripheral lymphoid organs and natural killer cells depends on the helix-loop-helix inhibitor Id2.

Transcription factors with a basic helix–loop–helix (HLH) motif have been shown to be crucial for various cell differentiation processes during development of multicellular organisms. Id proteins inhibit the functions of these transcription factors in a dominant-negative manner by suppressing their heterodimerization partners through the HLH domains. Members of the Id family also promote cell proliferation, implying a role in the control of cell differentiation. Here we show that Id2 is indispensable for normal development of mice. Id2−/− mice lack lymph nodes and Peyers patches. However, their splenic architecture is normal, exhibiting T-cell and B-cell compartments and distinct germinal centres. The cell population that produces lymphotoxins, essential factors for the development of secondary lymphoid organs, is barely detectable in the Id2−/− intestine. Furthermore, the null mutants show a greatly reduced population of natural killer (NK) cells, which is due to an intrinsic defect in NK-cell precursors. Our results indicate that Id2 has an essential role in the generation of peripheral lymphoid organs and NK cells.

Regulation of humoral and cellular gut immunity by lamina propria dendritic cells expressing Toll-like receptor 5

The intestinal cell types responsible for defense against pathogenic organisms remain incompletely characterized. Here we identify a subset of CD11chiCD11bhi lamina propria dendritic cells (LPDCs) that expressed Toll-like receptor 5 (TLR5) in the small intestine. When stimulated by the TLR5 ligand flagellin, TLR5+ LPDCs induced the differentiation of naive B cells into immunoglobulin A–producing plasma cells by a mechanism independent of gut-associated lymphoid tissue. In addition, by a mechanism dependent on TLR5 stimulation, these LPDCs promoted the differentiation of antigen-specific interleukin 17–producing T helper cells and type 1 T helper cells. Unlike spleen DCs, the LPDCs specifically produced retinoic acid, which, in a dose-dependent way, supported the generation and retention of immunoglobulin A–producing cells in the lamina propria and positively regulated the differentiation interleukin 17–producing T helper cells. Our findings demonstrate unique properties of LPDCs and the importance of TLR5 for adaptive immunity in the intestine.

Transcriptional profiling identifies Id2 function in dendritic cell development

Dendritic cells (DCs) are potent antigen-presenting cells with a pivotal role in antigen-specific immune responses. Here, we found that the helix-loop-helix transcription factor Id2 is up-regulated during DC development in vitro and crucial for the development of distinct DC subsets in vivo. Id2−/− mice lack Langerhans cells (LCs), the cutaneous contingent of DCs, and the splenic CD8α+ DC subset is markedly reduced. Mice deficient for transforming growth factor (TGF)-β also lack LCs, and we demonstrate here that, in DCs, TGF-β induces Id2 expression. We also show that Id2 represses B cell genes in DCs. These findings reveal a TGF-β–Id2 signaling pathway in DCs and suggest a mechanism by which Id2 affects the lineage choice of B cell and DC progenitors.

Mature natural killer cell and lymphoid tissue–inducing cell development requires Id2-mediated suppression of E protein activity

The Id2 transcriptional repressor is essential for development of natural killer (NK) cells, lymphoid tissue–inducing (LTi) cells, and secondary lymphoid tissues. Id2 was proposed to regulate NK and LTi lineage specification from multipotent progenitors through suppression of E proteins. We report that NK cell progenitors are not reduced in the bone marrow (BM) of Id2−/− mice, demonstrating that Id2 is not essential for NK lineage specification. Rather, Id2 is required for development of mature (m) NK cells. We define the mechanism by which Id2 functions by showing that a reduction in E protein activity, through deletion of E2A, overcomes the need for Id2 in development of BM mNK cells, LTi cells, and secondary lymphoid tissues. However, mNK cells are not restored in the blood or spleen of Id2−/−E2A−/− mice, suggesting a role for Id2 in suppression of alternative E proteins after maturation. Interestingly, the few splenic mNK cells in Id2−/− and Id2−/−E2A−/− mice have characteristics of thymus-derived NK cells, which develop in the absence of Id2, implying a differential requirement for Id2 in BM and thymic mNK development. Our findings redefine the essential functions of Id2 in lymphoid development and provide insight into the dynamic regulation of E and Id proteins during this process.

The Cytokine RANKL Produced by Positively Selected Thymocytes Fosters Medullary Thymic Epithelial Cells that Express Autoimmune Regulator

The thymic medulla provides a microenvironment where medullary thymic epithelial cells (mTECs) express autoimmune regulator and diverse tissue-restricted genes, contributing to launching self-tolerance. Positive selection is essential for thymic medulla formation via a previously unknown mechanism. Here we show that the cytokine RANK ligand (RANKL) was produced by positively selected thymocytes and regulated the cellularity of mTEC by interacting with RANK and osteoprotegerin. Forced expression of RANKL restored thymic medulla in mice lacking positive selection, whereas RANKL perturbation impaired medulla formation. These results indicate that RANKL produced by positively selected thymocytes is responsible for fostering thymic medulla formation, thereby establishing central tolerance.

Role of Id family proteins in growth control

Id proteins (inhibitors of DNA binding/differentiation) are negative regulators of basic helix‐loop‐helix (bHLH) type transcription factors, which promote the differentiation of various cell types. In addition to their “classical” ability to inhibit cell differentiation, they are able to stimulate cell cycle progression. These facts suggest that Id proteins play a role in keeping precursor cells immature and in expanding the cell population size during development. In vitro as well as in vivo analyses in the last several years have shown that Id proteins have more complex activities; they induce apoptosis or function as survival factors, depending on the cell context. Furthermore, dysregulated expression of Id proteins has been reported in several human tumors and seems to be related to the malignant character of tumors. Here, we summarize and discuss the biological activities of Id proteins from the standpoint of cell growth control. J. Cell. Physiol. 190: 21–28, 2002.

The Balance Between Pax5 and Id2 Activities Is the Key to AID Gene Expression

Pax5 activity is enhanced in activated B cells and is essential for class switch recombination (CSR). We show that inhibitor of differentiation (Id)2 suppresses CSR by repressing the gene expression of activation-induced cytidine deaminase (AID), which has been shown to be indispensable for CSR. Furthermore, a putative regulatory region of AID contains E2A- and Pax5-binding sites, and the latter site is indispensable for AID gene expression. Moreover, the DNA-binding activity of Pax5 is decreased in Id2-overexpressing B cells and enhanced in Id2−/− B cells. The kinetics of Pax5, but not E2A, occupancy to AID locus is the same as AID expression in primary B cells. Finally, enforced expression of Pax5 induces AID transcription in pro–B cell lines. Our results provide evidence that the balance between Pax5 and Id2 activities has a key role in AID gene expression.

Id and development.

During development, it is obvious that enormous multiplication and diversification of cells is required to build a body plan from a single fertilized egg and that these two processes, proliferation and differentiation, must be coordinated properly. Id proteins, negative regulators of basic helix–loop–helix transcription factors, possess the ability to inhibit differentiation and to stimulate proliferation, and are useful molecules for investigating the mechanisms regulating development. In the past few years, our understanding of the roles of Id proteins has been substantially enhanced by the detailed investigation of genetically modified animals. The data have indicated that the functions of Id proteins in vivo are functionally related to those revealed by earlier work in cell culture systems. However, unexpected organs and cell types have also been found to require Id proteins for their normal development. This review looks at the advances made in our understanding of the in vivo functions of Id proteins. The topics discussed include neurogenesis, natural killer cell development, lymphoid organogenesis, mammary gland development and spermatogenesis.

A Molecular Pathway Including Id2, Tbx5, and Nkx2-5 Required for Cardiac Conduction System Development

The cardiac conduction system is an anatomically discrete segment of specialized myocardium that initiates and propagates electrical impulses to coordinate myocardial contraction. To define the molecular composition of the mouse ventricular conduction system we used microdissection and transcriptional profiling by serial analysis of gene expression (SAGE). Conduction-system-specific expression for Id2, a member of the Id gene family of transcriptional repressors, was identified. Analyses of Id2-deficient mice demonstrated structural and functional conduction system abnormalities, including left bundle branch block. A 1.2 kb fragment of the Id2 promoter proved sufficient for cooperative regulation by Nkx2-5 and Tbx5 in vitro and for conduction-system-specific gene expression in vivo. Furthermore, compound haploinsufficiency of Tbx5 and Nkx2-5 or Tbx5 and Id2 prevented embryonic specification of the ventricular conduction system. We conclude that a molecular pathway including Tbx5, Nkx2-5, and Id2 coordinates specification of ventricular myocytes into the ventricular conduction system lineage.

Initiation of NALT organogenesis is independent of the IL-7R, LTβR, and NIK signaling pathways but requires the Id2 gene and CD3-CD4+CD45+ cells

Initiation of nasopharyngeal-associated lymphoid tissue (NALT) development is independent of the programmed cytokine cascade necessary for the formation of Peyers patches (PP) and peripheral lymph nodes (PLN), a cytokine cascade which consists of IL-7R, LTalpha1beta2/LTbetaR, and NIK. However, the subsequent organization of NALT seems to be controlled by these cytokine signaling cascades since the maturation of NALT structure is generally incomplete in those cytokine cascade-deficient mice. NALT as well as PP and PLN are completely absent in Id2(-/-) mice. NALT organogenesis is initiated following the adoptive transfer of CD3(-)CD4(+)CD45(+) cells into Id2(-/-) mice, constituting direct evidence that CD3(-)CD4(+)CD45(+) inducer cells can provide an IL-7R-, LTalpha1beta2/LTbetaR-, and NIK-independent tissue organogenesis pathway for secondary lymphoid tissue development.