The critical role of Rap1-GAPs Rasa3 and Sipa1 in T cells for pulmonary transit and egress from the lymph nodes.

Rap1-GTPase activates integrins and plays an indispensable role in lymphocyte trafficking, but the importance of Rap1 inactivation in this process remains unknown. Here we identified the Rap1-inactivating proteins Rasa3 and Sipa1 as critical regulators of lymphocyte trafficking. The loss of Rasa3 and Sipa1 in T cells induced spontaneous Rap1 activation and adhesion. As a consequence, T cells deficient in Rasa3 and Sipa1 were trapped in the lung due to firm attachment to capillary beds, while administration of LFA1 antibodies or loss of talin1 or Rap1 rescued lung sequestration. Unexpectedly, mutant T cells exhibited normal extravasation into lymph nodes, fast interstitial migration, even greater chemotactic responses to chemokines and sphingosine-1-phosphate, and entrance into lymphatic sinuses but severely delayed exit: mutant T cells retained high motility in lymphatic sinuses and frequently returned to the lymph node parenchyma, resulting in defective egress. These results reveal the critical trafficking processes that require Rap1 inactivation.

Thymocyte Development of Humanized Mice Is Promoted by Interactions with Human-Derived Antigen Presenting Cells upon Immunization.

Immune responses in humanized mice are generally inefficient without co-transplantation of human thymus or HLA transgenes. Previously, we generated humanized mice via the intra-bone marrow injection of CD133+ cord blood cells into irradiated adult immunodeficient mice (IBMI-huNSG mice), which could mount functional immune responses against HTLV-1, although the underlying mechanisms were still unknown. Here, we investigated thymocyte development in IBMI-huNSG mice, focusing on the roles of human and mouse MHC restriction. IBMI-huNSG mice had normal developmental profiles but aberrant thymic structures. Surprisingly, the thymic medulla-like regions expanded after immunization due to enhanced thymocyte expansion in association with the increase in HLA-DR+ cells, including CD205+ dendritic cells (DCs). The organ culture of thymus from immunized IBMI-huNSG mice with a neutralizing antibody to HLA-DR showed the HLA-DR-dependent expansion of CD4 single positive thymocytes. Mature peripheral T-cells exhibited alloreactive proliferation when co-cultured with human peripheral blood mononuclear cells. Live imaging of the thymus from immunized IBMI-huNSG mice revealed dynamic adhesive contacts of human-derived thymocytes and DCs accompanied by Rap1 activation. These findings demonstrate that an increase in HLA-DR+ cells by immunization promotes HLA-restricted thymocyte expansion in humanized mice, offering a unique opportunity to generate humanized mice with ease.

Rap1 organizes lymphocyte front-back polarity via RhoA signaling and talin1.

Lymphocyte trafficking requires fine-tuning of chemokine-mediated cell migration. This process depends on cytoskeletal dynamics and polarity, but its regulation remains elusive. We quantitatively measured cell polarity and revealed critical roles performed by integrin activator Rap1 in this process, independent of substrate adhesion. Rap1-deficient naive T cells exhibited impaired abilities to reorganize the actin cytoskeleton into pseudopods and actomyosin-rich uropods. Rap1-GTPase activating proteins (GAPs), Rasa3 and Sipa1, maintained an unpolarized shape; deletion of these GAPs spontaneously induced cell polarization, indicative of the polarizing effect of Rap1. Rap1 activation required F-actin scaffolds, and stimulated RhoA activation and actomyosin contractility at the rear. Furthermore, talin1 acted on Rap1 downstream effectors to promote actomyosin contractility in the uropod, which occurred independently of substrate adhesion and talin1 binding to integrins. These findings indicate that Rap1 signaling to RhoA and talin1 regulates chemokine-stimulated lymphocyte polarization and chemotaxis in a manner independent of adhesion.

Distinct bidirectional regulation of LFA1 and α4ß7 by Rap1 and integrin adaptors in T cells under shear flow.

Bidirectional control of integrin activation plays crucial roles in cell adhesive behaviors, but how integrins are specifically regulated by inside-out and outside-in signaling has not been fully understood. Here, we report distinct bidirectional regulation of major lymphocyte homing receptors LFA1 and α4ß7 in primary T cells. A small increase of Rap1 activation in L-selectin-mediated tether/rolling was boosted by the outside-in signaling from ICAM1-interacting LFA1 through subsecond, simultaneous activation of Rap1 GTPase and talin1, but not kindlin-3, resulting in increased capture and slowing. In contrast, none of them were required for tether/rolling by α4ß7 on MAdCAM1. High Rap1 activation with chemokines or the loss of Rap1-inactivating proteins Rasa3 and Sipa1 increased talin1/kindlin-3-dependent arrest with high-affinity binding of LFA1 to membrane-anchored ICAM1. However, despite increased affinity of α4ß7, activated Rap1 severely suppressed adhesion on MAdCAM1 under shear flow, indicating the critical importance of a sequential outside-in/inside-out signaling for α4ß7.

LFA1 Activation: Insights from a Single-Molecule Approach.

Integrin LFA1 is a cell adhesion receptor expressed exclusively in leukocytes, and plays crucial roles in lymphocyte trafficking, antigen recognition, and effector functions. Since the discovery that the adhesiveness of LFA1 can be dynamically changed upon stimulation, one challenge has been understanding how integrins are regulated by inside-out signaling coupled with macromolecular conformational changes, as well as ligand bindings that transduce signals from the extracellular domain to the cytoplasm in outside-in signaling. The small GTPase Rap1 and integrin adaptor proteins talin1 and kindlin-3 have been recognized as critical molecules for integrin activation. However, their cooperative regulation of integrin adhesiveness in lymphocytes requires further research. Recent advances in single-molecule imaging techniques have revealed dynamic molecular processes in real-time and provided insight into integrin activation in cellular environments. This review summarizes integrin regulation and discusses new findings regarding the bidirectionality of LFA1 activation and signaling processes in lymphocytes.

Kindlin-3 disrupts an intersubunit association in the integrin LFA1 to trigger positive feedback activation by Rap1 and talin1.

Integrin activation by the intracellular adaptor proteins talin1 and kindlin-3 is essential for lymphocyte adhesion. These adaptors cooperatively control integrin activation through bidirectional (inside-out and outside-in) activation signals. Using single-molecule measurements, we revealed the distinct dynamics of talin1 and kindlin-3 interactions with the integrin LFA1 (αLß2) and their functions in LFA1 activation and LFA1-mediated adhesion. The kinetics of talin1 binding to the tail of the ß2 subunit corresponded to those of LFA1 binding to its ligand ICAM1. ICAM1 binding induced transient interactions between the membrane-proximal cytoplasmic region of the ß2 subunit with an N-terminal domain of kindlin-3, leading to disruption of the association between the integrin subunits (the α/ß clasp) and unbending of the ectodomains of the α/ß heterodimer. These conformational changes promoted high-affinity talin1 binding to the ß2 tail that required the talin rod domain and the actomyosin cytoskeleton. Inside-out signaling induced by the GTPase Rap1 did not markedly stabilize the binding of talin1 and kindlin-3 to LFA1. In contrast, ligand-induced outside-in signaling, the stabilization of open LFA1 conformers, or shear force substantially altered the dynamics of talin1 and kindlin-3 association with LFA1 and enhanced both Rap1 and LFA1 activation. In migrating lymphocytes, asymmetrical distribution of talin1 and kindlin-3 correlated with the maturation of LFA1 from a low-affinity conformation at the leading edge to a high-affinity conformation in the adherent mid-body. Our results suggest that kindlin-3 spatiotemporally mediates a positive feedback circuit of LFA1 activation to control dynamic adhesion and migration of lymphocytes.

Effect of Learning Dataset for Identification of Active Molecules: A Case Study of Integrin αIIbß3 Inhibitors.

Efficient in silico approaches are needed to identify strong integrin αIIbß3 inhibitors through a small number of measurements. To address the challenge, we investigated the effect of learning dataset on the classification performance of machine learning models focusing on weak and inactive compounds. The structure and activity information of the compounds were obtained from ChEMBL, and pCHEMBL values were used to classify them as active, inactive, or weak. Datasets with various imbalance levels from active:inactive=1 : 1 to 1 : 1000 were used for the machine learning. The prediction scores of the weak samples were found to lie between the predictive values of active and inactive compounds. In addition, another dataset that consists of 149 actives and 6.9 million inactives was screened; the results indicated that the number of positive predictions decreased for models trained with a higher number of inactives. Although there is a trade-off between false positives and false negatives, for determination of compounds with strong activity using a reduced number of measurements, it is better to use a large number of inactives for learning and identifying compounds that score higher than the weak samples.

MST1/2 Balance Immune Activation and Tolerance by Orchestrating Adhesion, Transcription, and Organelle Dynamics in Lymphocytes.

The STE20-like serine/threonine kinases MST1 and MST2 (MST1/2) are mammalian homologs of Hippo in flies. MST1/2 regulate organ size by suppressing the transcription factor YAP, which promotes proliferation. MST1 is predominantly expressed in immune cells, where it plays distinct roles. Here, we review the functions of MST1/2 in immune cells, uncovered by a series of recent studies, and discuss the connection between MST1/2 function and immune responses. MST1/2 regulate lymphocyte development, trafficking, survival, and antigen recognition by naive T cells. MST1/2 also regulate the function of regulatory T cells and effector T cell differentiation, thus acting to balance immune activation and tolerance. Interestingly, MST1/2 elicit these functions not by the "canonical" Hippo pathway, but by the non-canonical Hippo pathway or alternative pathways. In these pathways, MST1/2 regulates cellular processes relating to immune response, such as chemotaxis, cell adhesion, immunological synapse, gene transcriptions. Recent advances in our understanding of the molecular mechanisms of these processes have revealed important roles of MST1/2 in regulating cytoskeleton remodeling, integrin activation, and vesicular transport in lymphocytes. We discuss the significance of the MST1/2 signaling in lymphocytes in the regulation of organelle dynamics.

Live-Cell FRET Imaging Reveals a Role of Extracellular Signal-Regulated Kinase Activity Dynamics in Thymocyte Motility.

Extracellular signal-regulated kinase (ERK) plays critical roles in T cell development in the thymus. Nevertheless, the dynamics of ERK activity and the role of ERK in regulating thymocyte motility remain largely unknown due to technical limitations. To visualize ERK activity in thymocytes, we here developed knockin reporter mice expressing a Förster/fluorescence resonance energy transfer (FRET)-based biosensor for ERK from the ROSA26 locus. Live imaging of thymocytes isolated from the reporter mice revealed that ERK regulates thymocyte motility in a subtype-specific manner. Negative correlation between ERK activity and motility was observed in CD4/CD8 double-positive thymocytes and CD8 single-positive thymocytes, but not in CD4 single-positive thymocytes. Interestingly, however, the temporal deviations of ERK activity from the average correlate with the motility of CD4 single-positive thymocytes. Thus, live-cell FRET imaging will open a window to understanding the dynamic nature and the diverse functions of ERK signaling in T cell biology.

Essential Role of Canonical NF-κB Activity in the Development of Stromal Cell Subsets in Secondary Lymphoid Organs.

Organized tissue structure in the secondary lymphoid organs (SLOs) tightly depends on the development of fibroblastic stromal cells (FSCs) of mesenchymal origin; however, the mechanisms of this relationship are poorly understood. In this study, we specifically inactivated the canonical NF-κB pathway in FSCs in vivo by conditionally inducing IκBα mutant in a Ccl19-IκBSR mouse system in which NF-κB activity is likely to be suppressed in fetal FSC progenitors. Given that NF-κB activation in fetal FSCs is essential for SLO development, the animals were expected to lack SLOs. However, all SLOs were preserved in Ccl19-IκBSR mice. Instead, the T cell area was severely disturbed by the lack of CCL21-expressing FSCs, whereas the follicles and associated FSC networks were formed. Fate mapping revealed that IκBSR-expressing cells constituted only a small fraction of stromal compartment outside the follicles. Taken together, our findings indicate an essential role of the canonical NF-κB pathway activity in the development of three FSC subsets common to SLOs and suggest transient or stochastic CCL19 expression in FSC progenitors and a compensatory differentiation program of follicular FSCs.

Visualizing the Rapid and Dynamic Elimination of Allogeneic T Cells in Secondary Lymphoid Organs.

Allogeneic organ transplants are rejected by the recipient immune system within several days or weeks. However, the rejection process of allogeneic T (allo-T) cells is poorly understood. In this study, using fluorescence-based monitoring and two-photon live imaging in mouse adoptive transfer system, we visualized the fate of allo-T cells in the in vivo environment and showed rapid elimination in secondary lymphoid organs (SLOs). Although i.v. transferred allo-T cells efficiently entered host SLOs, including lymph nodes and the spleen, ∼70% of the cells had disappeared within 24 h. At early time points, allo-T cells robustly migrated in the T cell area, whereas after 8 h, the numbers of arrested cells and cell fragments were dramatically elevated. Apoptotic breakdown of allo-T cells released a large amount of cell debris, which was efficiently phagocytosed and cleared by CD8+ dendritic cells. Rapid elimination of allo-T cells was also observed in nu/nu recipients. Depletion of NK cells abrogated allo-T cell reduction only in a specific combination of donor and recipient genetic backgrounds. In addition, F1 hybrid transfer experiments showed that allo-T cell killing was independent of the missing-self signature typically recognized by NK cells. These suggest the presence of a unique and previously uncharacterized modality of allorecognition by the host immune system. Taken together, our findings reveal an extremely efficient and dynamic process of allogeneic lymphocyte elimination in SLOs, which could not be recapitulated in vitro and is distinct from the rejection of solid organ and bone marrow transplants.

Mode of Tolerance Induction and Requirement for Aire Are Governed by the Cell Types That Express Self-Antigen and Those That Present Antigen.

Aire controls the fate of autoreactive thymocytes (i.e., clonal deletion or development into regulatory T cells [Tregs]) through transcriptional control of the expression of tissue-restricted self-antigens (TRAs) from medullary thymic epithelial cells (mTECs) and bone marrow (BM)-derived cells. Although TRAs expressed by mTECs and BM-derived cells are suggested to complement each other to generate a full spectrum of TRAs, little is known about the relative contribution of TRAs from each component for establishment of self-tolerance. Furthermore, the precise role of Aire in specific types of Aire-expressing APCs remains elusive. We have approached these issues by generating two different types of transgenic mouse (Tg) model, which express a prefixed model self-antigen driven by the insulin promoter or the Aire promoter. In the insulin-promoter Tg model, mTECs alone were insufficient for clonal deletion, and BM-derived APCs were required for this action by utilizing Ag transferred from mTECs. In contrast, mTECs alone were able to induce Tregs, although at a much lower efficiency in the absence of BM-derived APCs. Importantly, lack of Aire in mTECs, but not in BM-derived APCs, impaired both clonal deletion and production of Tregs. In the Aire-promoter Tg model, both mTECs and BM-derived APCs could independently induce clonal deletion without Aire, and production of Tregs was impaired by the lack of Aire in mTECs, but not in BM-derived APCs. These results suggest that the fate of autoreactive thymocytes together with the requirement for Aire depend on the cell types that express self-antigens and the types of APCs involved in tolerance induction.

NDR1-Dependent Regulation of Kindlin-3 Controls High-Affinity LFA-1 Binding and Immune Synapse Organization.

Antigen-specific adhesion between T cells and antigen-presenting cells (APC) during the formation of the immunological synapse (IS) is mediated by LFA-1 and ICAM-1. Here, LFA-1-ICAM-1 interactions were measured at the single-molecule level on supported lipid bilayers. High-affinity binding was detected at low frequencies in the inner peripheral supramolecular activation cluster (SMAC) zone that contained high levels of activated Rap1 and kindlin-3. Rap1 was essential for T cell attachment, whereas deficiencies of ste20-like kinases, Mst1/Mst2, diminished high-affinity binding and abrogated central SMAC (cSMAC) formation with mislocalized kindlin-3 and vesicle transport regulators involved in T cell receptor recycling/releasing machineries, resulting in impaired T cell-APC interactions. We found that NDR1 kinase, activated by the Rap1 signaling cascade through RAPL and Mst1/Mst2, associated with and recruited kindlin-3 to the IS, which was required for high-affinity LFA-1/ICAM-1 binding and cSMAC formation. Our findings reveal crucial roles for Rap1 signaling via NDR1 for recruitment of kindlin-3 and IS organization.

Microenvironmental Control of High-Speed Interstitial T Cell Migration in the Lymph Node.

T cells are highly concentrated in the lymph node (LN) paracortex, which serves an important role in triggering adoptive immune responses. Live imaging using two-photon laser scanning microscopy revealed vigorous and non-directional T cell migration within this area at average velocity of more than 10 µm/min. Active interstitial T cell movement is considered to be crucial for scanning large numbers of dendritic cells (DCs) to find rare cognate antigens. However, the mechanism by which T cells achieve such high-speed movement in a densely packed, dynamic tissue environment is not fully understood. Several new findings suggest that fibroblastic reticular cells (FRCs) and DCs control T cell movement in a multilateral manner. Chemokines and lysophosphatidic acid produced by FRCs cooperatively promote the migration, while DCs facilitate LFA-1-dependent motility via expression of ICAM-1. Furthermore, the highly dense and confined microenvironment likely plays a key role in anchorage-independent motility. We propose that T cells dynamically switch between two motility modes; anchorage-dependent and -independent manners. Unique tissue microenvironment and characteristic migration modality of T cells cooperatively generate high-speed interstitial movement in the LN.

The RacGAP protein FilGAP is a negative regulator of chemokine-promoted lymphocyte migration.

Rho family small GTPases regulate lymphocyte migration induced by chemokines. However, how lymphocyte migration is regulated by Rho GTPases remains to be elucidated. Here, we identified FilGAP, a Rac-specific GAP, as a negative regulator of lymphocyte polarization and migration. Depletion of FilGAP in mouse pro-B BAF cells increased cellular elongation and membrane protrusion after stimulation of the cells with SDF-1α, which caused increased migration speed. Although FilGAP is detectable both at the front and rear of polarized cells, FilGAP appears to be concentrated at the tip of retracting lamellae of moving lymphocytes. Moreover, depletion of FilGAP increased activation of Rac at the front of polarized cells. Thus, FilGAP may inhibit lamellae extension at the front of moving lymphocytes.

Sema3e/Plexin D1 Modulates Immunological Synapse and Migration of Thymocytes by Rap1 Inhibition.

Regulation of thymocyte trafficking plays an important role during thymic selection, but our understanding of the molecular mechanisms underlying these processes is limited. In this study, we demonstrated that class III semaphorin E (sema3e), a guidance molecule during neural and vascular development, directly inhibited Rap1 activation and LFA-1-dependent adhesion through the GTPase-activating protein activity of plexin D1. Sema3e inhibited Rap1 activation of thymocytes in response to chemokines and TCR stimulation, LFA-mediated adhesion, and T cell-APC interactions. Immunological synapse (IS) formation in mature thymocytes on supported lipid bilayers was also attenuated by sema3e. Impaired IS formation was associated with reduced Rap1 activation on the contact surface and cell periphery. Moreover, a significant increase of CD4(+) thymocytes was detected in the medulla of mice with T cell lineage-specific deletion of plexin D1. Two-photon live imaging of thymic explants and slices revealed enhanced Rap1 activation and migration of CD69(+) double-positive and single-positive cells with plexin D1 deficiency. Our results demonstrate that sema3e/plexin D1 modulates IS formation and Ag-scanning activities of thymocytes within thymic tissues.

Enhanced cytotoxic T-cell function and inhibition of tumor progression by Mst1 deficiency.

Mammalian ste-20 like kinase Mst1 plays important roles during apoptosis, proliferation, cell polarity, and migration. Here, we report a novel role of Mst1 for cytotoxic T-cell responses and tumor suppression. The defect of Mst1 caused decreased levels of FoxO, and promoted cytotoxicity in vitro. Mst1(-/-) cytotoxic T cells also exhibited enhanced T-bet expression that was associated with elevated expression levels of IFNγ and granzyme B. Moreover, Mst1(-/-) cytotoxic T cells suppressed tumor growth in vivo. The data suggest that Mst1 inhibits cytotoxicity via T-bet suppression by FoxO1 and FoxO3a. Thus, Mst1 is a potential therapeutic target for tumor immunotherapy.

Integrin LFA-1 regulates cell adhesion via transient clutch formation.

Integrin LFA-1 regulates immune cell adhesion and trafficking by binding to ICAM-1 upon chemokine stimulation. Integrin-mediated clutch formation between extracellular ICAM-1 and the intracellular actin cytoskeleton is important for cell adhesion. We applied single-molecule tracking analysis to LFA-1 and ICAM-1 in living cells to examine the ligand-binding kinetics and mobility of the molecular clutch under chemokine-induced physiological adhesion and Mn(2+)-induced tight adhesion. Our results show a transient LFA-1-mediated clutch formation that lasts a few seconds and leads to a transient lower-mobility is sufficient to promote cell adhesion. Stable clutch formation was observed for Mn(2+)-induced high affinity LFA-1, but was not required for physiological adhesion. We propose that fast cycling of the clutch formation by intermediate-affinity integrin enables dynamic cell adhesion and migration.

Hypermethylation of MST1 in IgG4-related autoimmune pancreatitis and rheumatoid arthritis.

The serine/threonine kinase Mst1 plays important roles in the control of immune cell trafficking, proliferation, and differentiation. Previously, we reported that Mst1 was required for thymocyte selection and regulatory T-cell functions, thereby the prevention of autoimmunity in mice. In humans, MST1 null mutations cause T-cell immunodeficiency and hypergammaglobulinemia with autoantibody production. RASSF5C(RAPL) is an activator of MST1 and it is frequently methylated in some tumors. Herein, we investigated methylation of the promoter regions of MST1 and RASSF5C(RAPL) in leukocytes from patients with IgG4-related autoimmune pancreatitis (AIP) and rheumatoid arthritis (RA). Increased number of CpG methylation in the 5' region of MST1 was detected in AIP patients with extrapancreatic lesions, whereas AIP patients without extrapancreatic lesions were similar to controls. In RA patients, we detected a slight increased CpG methylation in MST1, although the overall number of methylation sites was lower than that of AIP patients with extrapancreatic lesions. There were no significant changes of the methylation levels of the CpG islands in the 5' region of RASSF5C(RAPL) in leukocytes from AIP and RA patients. Consistently, we found a significantly down-regulated expression of MST1 in regulatory T cells of AIP patients. Our results suggest that the decreased expression of MST1 in regulatory T cells due to hypermethylation of the promoter contributes to the pathogenesis of IgG4-related AIP.