Tomoya Katakai

Lymph Node Fibroblastic Reticular Cells Construct the Stromal Reticulum via Contact with Lymphocytes

The sophisticated microarchitecture of the lymph node, which is largely supported by a reticular network of fibroblastic reticular cells (FRCs) and extracellular matrix, is essential for immune function. How FRCs form the elaborate network and remodel it in response to lymphocyte activation is not understood. In this work, we established ERTR7+gp38+VCAM-1+ FRC lines and examined the production of the ER-TR7 antigen. Multiple chemokines produced by FRCs induced T cell and dendritic cell chemotaxis and adhesion to the FRC surface. FRCs can secrete the ER-TR7 antigen as an extracellular matrix component to make a reticular meshwork in response to contact with lymphocytes. The formation of the meshwork is induced by stimulation with tumor necrosis factor-α or lymphotoxin-α in combination with agonistic antibody to lymphotoxin-β receptor in a nuclear factor-κB (RelA)–dependent manner. These findings suggest that signals from lymphocytes induce FRCs to form the network that supports the movement and interactions of immune effectors within the lymph node.

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.

Organizer-Like Reticular Stromal Cell Layer Common to Adult Secondary Lymphoid Organs

Mesenchymal stromal cells are crucial components of secondary lymphoid organs (SLOs). Organogenesis of SLOs involves specialized stromal cells, designated lymphoid tissue organizer (LTo) in the embryonic anlagen; in the adult, several distinct stromal lineages construct elaborate tissue architecture and regulate lymphocyte compartmentalization. The relationship between the LTo and adult stromal cells, however, remains unclear, as does the precise number of stromal cell types that constitute mature SLOs are unclear. From mouse lymph nodes, we established a VCAM-1+ICAM-1+MAdCAM-1+ reticular cell line that can produce CXCL13 upon LTβR stimulation and support primary B cell adhesion and migration in vitro. A similar stromal population sharing many characteristics with the LTo, designated marginal reticular cells (MRCs), was found in the outer follicular region immediately underneath the subcapsular sinus of lymph nodes. Moreover, MRCs were commonly observed at particular sites in various SLOs even in Rag2−/− mice, but were not found in ectopic lymphoid tissues, suggesting that MRCs are a developmentally determined element. These findings lead to a comprehensive view of the stromal composition and architecture of SLOs.

Roles of p-ERM and Rho–ROCK signaling in lymphocyte polarity and uropod formation

Front–rear asymmetry in motile cells is crucial for efficient directional movement. The uropod in migrating lymphocytes is a posterior protrusion in which several proteins, including CD44 and ezrin/radixin/moesin (ERM), are concentrated. In EL4.G8 T-lymphoma cells, Thr567 phosphorylation in the COOH-terminal domain of ezrin regulates the selective localization of ezrin in the uropod. Overexpression of the phosphorylation-mimetic T567D ezrin enhances uropod size and cell migration. T567D ezrin also induces construction of the CD44-associated polar cap, which covers the posterior cytoplasm in staurosporine-treated, uropod-disrupted EL4.G8 cells or in naturally unpolarized X63.653 myeloma cells in an actin cytoskeleton–dependent manner. Rho-associated coiled coil–containing protein kinase (ROCK) inhibitor Y-27632 disrupts the uropod but not the polar cap, indicating that Rho–ROCK signaling is required for posterior protrusion but not for ERM phosphorylation. Phosphorylated ezrin associates with Dbl through its NH2-terminal domain and causes Rho activation. Moreover, constitutively active Q63L RhoA is selectively localized in the rear part of the cells. Thus, phosphorylated ERM has a potential function in establishing plasma membrane “posteriority” in the induction of the uropod in T lymphocytes.

Essential role of Id2 in negative regulation of IgE class switching.

Serum concentrations of immunoglobulin E (IgE) in normal circumstances are kept much lower than those of other Ig isotypes to avoid allergic reactions. B cells lacking Id2 have increased E2A activity, which leads to specific enhancement of germline transcription of the immunoglobulin ε locus. As a consequence, Id2-deficient B cells undergo class switch recombination (CSR) to IgE at a much higher frequency than wild-type B cells. In contrast, Id2 is induced in wild-type B cells by transforming growth factor-β1 (TGF-β1) and suppresses IgE CSR. Our results provide evidence for the inhibitory and selective role of Id2 in IgE CSR in response to TGF-β1. Id2 might act as molecular safeguard to suppress IgE CSR to prevent serious complications such as allergic hypersensitivity during the normal course of immune responses.

DOCK8 is a Cdc42 activator critical for interstitial dendritic cell migration during immune responses

To migrate efficiently through the interstitium, dendritic cells (DCs) constantly adapt their shape to the given structure of the extracellular matrix and follow the path of least resistance. It is known that this amoeboid migration of DCs requires Cdc42, yet the upstream regulators critical for localization and activation of Cdc42 remain to be determined. Mutations of DOCK8, a member of the atypical guanine nucleotide exchange factor family, causes combined immunodeficiency in humans. In the present study, we show that DOCK8 is a Cdc42-specific guanine nucleotide exchange factor that is critical for interstitial DC migration. By generating the knockout mice, we found that in the absence of DOCK8, DCs failed to accumulate in the lymph node parenchyma for T-cell priming. Although DOCK8-deficient DCs migrated normally on 2-dimensional surfaces, DOCK8 was required for DCs to crawl within 3-dimensional fibrillar networks and to transmigrate through the subcapsular sinus floor. This function of DOCK8 depended on the DHR-2 domain mediating Cdc42 activation. DOCK8 deficiency did not affect global Cdc42 activity. However, Cdc42 activation at the leading edge membrane was impaired in DOCK8-deficient DCs, resulting in a severe defect in amoeboid polarization and migration. Therefore, DOCK8 regulates interstitial DC migration by controlling Cdc42 activity spatially.

Mst1 controls lymphocyte trafficking and interstitial motility within lymph nodes

The regulation of lymphocyte adhesion and migration plays crucial roles in lymphocyte trafficking during immunosurveillance. However, our understanding of the intracellular signalling that regulates these processes is still limited. Here, we show that the Ste20‐like kinase Mst1 plays crucial roles in lymphocyte trafficking in vivo. Mst1−/− lymphocytes exhibited an impairment of firm adhesion to high endothelial venules, resulting in an inefficient homing capacity. In vitro lymphocyte adhesion cascade assays under physiological shear flow revealed that the stopping time of Mst1−/− lymphocytes on endothelium was markedly reduced, whereas their L‐selectin‐dependent rolling/tethering and transition to LFA‐1‐mediated arrest were not affected. Mst1−/− lymphocytes were also defective in the stabilization of adhesion through α4 integrins. Consequently, Mst1−/− mice had hypotrophic peripheral lymphoid tissues and reduced marginal zone B cells and dendritic cells in the spleen, and defective emigration of single positive thymocytes. Furthermore, Mst1−/− lymphocytes had impaired motility over lymph node‐derived stromal cells and within lymph nodes. Thus, our data indicate that Mst1 is a key enzyme involved in lymphocyte entry and interstitial migration.

Mst1 regulates integrin-dependent thymocyte trafficking and antigen recognition in the thymus.

Thymocyte trafficking has an important role in thymic selection. Here we show that the Hippo homologue Mst1 is required for thymocyte migration and antigen recognition by LFA-1 and ICAM-1 within the medulla. Using two-photon imaging of thymic tissues, we found that highly motile mature thymocytes arrest and are activated in the vicinity of rare populations of Aire(+) ICAM-1(hi) medullary thymic epithelia in a negatively selecting environment. Notably, Mst1 deficiency or blocking the cell adhesion molecules LFA-1 and ICAM-1 results in inefficient migration and antigen recognition of CD4(+) thymocytes within the medulla. Consistent with these defects, thymocyte selection is impaired in Mst1(-/-) mice, which display T cell-dependent inflammatory infiltrates in multiple organs and develop autoantibodies. Our results suggest that Mst1 has a key role in regulating thymocyte self-antigen recognition in the medulla.

Targeting of Krüppel-associated Box-containing Zinc-finger Proteins to Centromeric Heterochromatin : Implication for the Gene Silencing Mechanisms

Krüppel-associated box-containing zinc finger proteins (KRAB-ZFPs) repress transcription via functional interaction with the corepressor KRAB-associated protein-1 (KAP-1). KAP-1 directly interacts with heterochromatin protein 1 (HP1), a dose-dependent regulator of heterochromatin-mediated silencing. Here we show that two KRAB-ZFPs that we previously identified, KRAZ1 and KRAZ2, are targeted to foci of centromeric heterochromatin containing HP1α through the interaction with KAP-1. Centromeric targeting potential of KRAZ1 and KAP-1 is strictly correlated with their silencing activities; a KRAB mutant of KRAZ1 that is unable to bind KAP-1 and KAP-1 deletions unable to bind HP1 cannot localize to centromeric foci nor repress transcription. We provide evidence that this correlation is likely to be functionally relevant. First, overexpression of the VP16 transactivation domain fused with the KAP-1 deletion that binds to KRAB but not to HP1 leads to dramatic redistribution of KRAZ1 from centromeric foci and simultaneously converts KRAZ1-mediated silencing into strong transcriptional activation. Second, a specific inhibitor of histone deacetylases, trichostatin A, effectively redistributes KRAZ1 and KAP-1 from centromeric foci and partially relieves their silencing activities. These data strongly suggest that KRAB-ZFPs/KAP-1 silence transcription by dynamic recruitment of the target locus to the specific gene silencing compartment, centromeric heterochromatin, in a histone deacetylase-dependent manner.

Graded Attenuation of TCR Signaling Elicits Distinct Autoimmune Diseases by Altering Thymic T Cell Selection and Regulatory T Cell Function

Mice with a mutation of the ζ-associated protein of 70 kDa gene (skg mutation) are genetically prone to develop autoimmune arthritis, depending on the environment. In a set of mice with the mutation, the amount of ζ-associated protein of 70 kDa protein as well as its tyrosine phosphorylation upon TCR stimulation decreased from +/+, skg/+, skg/skg, to skg/− mice in a stepwise manner. The reduction resulted in graded alterations of thymic positive and negative selection of self-reactive T cells and Foxp3+ natural regulatory T cells (Tregs) and their respective functions. Consequently, skg/− mice spontaneously developed autoimmune arthritis even in a microbially clean environment, whereas skg/skg mice required stimulation through innate immunity for disease manifestation. After Treg depletion, organ-specific autoimmune diseases, especially autoimmune gastritis, predominantly developed in +/+, at a lesser incidence in skg/+, but not in skg/skg BALB/c mice, which suffered from other autoimmune diseases, especially autoimmune arthritis. In correlation with this change, gastritis-mediating TCR transgenic T cells were positively selected in +/+, less in skg/+, but not in skg/skg BALB/c mice. Similarly, on the genetic background of diabetes-prone NOD mice, diabetes spontaneously developed in +/+, at a lesser incidence in skg/+, but not in skg/skg mice, which instead succumbed to arthritis. Thus, the graded attenuation of TCR signaling alters the repertoire and the function of autoimmune T cells and natural Tregs in a progressive manner. It also changes the dependency of disease development on environmental stimuli. These findings collectively provide a model of how genetic anomaly of T cell signaling contributes to the development of autoimmune disease.