Molecular Imaging of PD-1 Unveils Unknown Characteristics of PD-1 Itself by Visualizing "PD-1 Microclusters".

Programmed cell death-1 (PD-1) is one of the most famous coinhibitory receptors that are expressed on effector T cells to regulate their function. The PD-1 ligands, PD-L1 and PD-L2, are expressed by various cells throughout the body at steady state and their expression was further regulated within different pathological conditions such as tumor-bearing and chronic inflammatory diseases. In recent years, immune checkpoint inhibitor (ICI) therapies with anti-PD-1 or anti-PD-L1 has become a standard treatment for various malignancies and has shown remarkable antitumor effects. Since the discovery of PD-1 in 1992, a huge number of studies have been conducted to elucidate the function of PD-1. Herein, this paper provides an overview of PD-1 biological findings and sheds some light on the current technology for molecular imaging of PD-1.

Evaluation of therapeutic PD-1 antibodies by an advanced single-molecule imaging system detecting human PD-1 microclusters.

With recent advances in immune checkpoint inhibitors (ICIs), immunotherapy has become the standard treatment for various malignant tumors. Their indications and dosages have been determined empirically, taking individually conducted clinical trials into consideration, but without a standard method to evaluate them. Here we establish an advanced imaging system to visualize human PD-1 microclusters, in which a minimal T cell receptor (TCR) signaling unit co-localizes with the inhibitory co-receptor PD-1 in vitro. In these microclusters PD-1 dephosphorylates both the TCR/CD3 complex and its downstream signaling molecules via the recruitment of a phosphatase, SHP2, upon stimulation with the ligand hPD-L1. In this system, blocking antibodies for hPD-1-hPD-L1 binding inhibits hPD-1 microcluster formation, and each therapeutic antibody (pembrolizumab, nivolumab, durvalumab and atezolizumab) is characterized by a proprietary optimal concentration and combinatorial efficiency enhancement. We propose that our imaging system could digitally evaluate PD-1-mediated T cell suppression to evaluate their clinical usefulness and to develop the most suitable combinations among ICIs or between ICIs and conventional cancer treatments.

Tas2R signaling enhances mouse neutrophil migration via a ROCK-dependent pathway.

Type-2 bitter taste receptors (Tas2Rs) are a large family of G protein-coupled receptors that are expressed in the oral cavity and serve to detect substances with bitter tastes in foods and medicines. Recent evidence suggests that Tas2Rs are also expressed extraorally, including in immune cells. However, the role of Tas2Rs in immune cells remains controversial. Here, we demonstrate that Tas2R126, Tas2R135, and Tas2R143 are expressed in mouse neutrophils, but not in other immune cells such as macrophages or T and B lymphocytes. Treatment of bone marrow-derived neutrophils from wild-type mice with the Tas2R126/143 agonists arbutin and d-salicin led to enhanced C-X-C motif chemokine ligand 2 (CXCL2)-stimulated migration in vitro, but this response was not observed in neutrophils from Tas2r126/135/143-deficient mice. Enhancement of CXCL2-stimulated migration by Tas2R agonists was accompanied by increased phosphorylation of myosin light chain 2 (MLC2) and was blocked by pretreatment of neutrophils with inhibitors of Rho-associated coiled-coil-containing protein kinase (ROCK), but not by inhibitors of the small GTPase RhoA. Taken together, these results demonstrate that mouse neutrophils express functional Tas2R126/143 and suggest a role for Tas2R126/143-ROCK-MLC2-dependent signaling in the regulation of neutrophil migration.

Micro- and Macro-Anatomical Frameworks of Lymph Nodes Indispensable for the Lymphatic System Filtering Function.

In the lymphatic vascular system, lymph nodes (LNs) play a pivotal role in filtering and removing lymph-borne substances. The filtering function of LNs involves resident macrophages tightly associated with unique lymphatic sinus structures. Moreover, an intermittently arranged LN in the lymphatic pathway is considered to cooperatively prevent lymph-borne substances from entering blood circulation. However, the functional significance of tissue microarchitecture, cellular composition, and individual LNs in the "LN chain" system is not fully understood. To explore the mechanistic and histo-anatomical significance of LNs as lymph fluid filters, we subcutaneously injected fluorescent tracers into mice and examined the details of lymphatic transport to the LNs qualitatively and quantitatively. Lymph-borne tracers were selectively accumulated in the MARCO+ subcapsular-medullary sinus border (SMB) region of the LN, in which reticular lymphatic endothelial cells and CD169+F4/80+ medullary sinus macrophages construct a dense meshwork of the physical barrier, forming the main body to capture the tracers. We also demonstrated stepwise filtration via the LN chain in the lymphatic basin, which prevented tracer leakage into the blood. Furthermore, inflammatory responses that induce the remodeling of LN tissue as well as the lymphatic pathway reinforce the overall filtering capacity of the lymphatic basin. Taken together, specialized tissue infrastructure in the LNs and their systematic orchestration constitute an integrated filtering system for lymphatic recirculation.

Lymph Node Stromal Cells: Diverse Meshwork Structures Weave Functionally Subdivided Niches.

Lymph nodes (LNs) are secondary lymphoid organs that function as the first line of defense against invasive foreign substances. Within the LNs, different types of immune cells are strategically localized to induce immune responses efficiently. Such a sophisticated tissue structure is a complex of functionally specialized niches, constructed by a variety of fibroblastic stromal cells. Elucidating the characteristics and functions of the niches and stromal cells will facilitate comprehension of the immune response induced in the LNs. Three recent studies offered novel insights into specialized stromal cells. In our discussion of these surprisingly diverse stromal cells, we will integrate information from these studies to improve knowledge about the structure and niches of LN.

Transdermal entry of yeast components elicits transient B cell-associated responses in skin-draining lymph nodes.

Immune responses to non-pathogenic yeasts induced within the draining lymph node remain to be understood. In this study, we have investigated the changes in lymphocytes and their activity in skin-draining lymph nodes in response to transdermally injected zymosan (component of the yeast cell wall). Zymosan elicited the transient increase of B cell number and activation status without affecting the capacity for proliferation. The increased B cell content in the regional lymph nodes was likely due to the reduction of B cell egress from the tissue and in part the increase of homing from the circulation. Zymosan also upregulated the inflammatory cytokines, such as IL-1ß, IL-6, IL-12, and IFNγ, regulatory cytokines IL-10 and TGFß, and lymphoid chemokine CXCL13. Among these, the expression of IL-12 and IL-10 was markedly high in B cells. Altogether, these findings demonstrate a unique B cell-associated response to non-pathogenic yeast component in the draining lymph nodes. This will provide insights into the clinical and healthcare applications of non-pathogenic beneficial microbes.

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.

A Distinct Subset of Fibroblastic Stromal Cells Constitutes the Cortex-Medulla Boundary Subcompartment of the Lymph Node.

The spatiotemporal regulation of immune responses in the lymph node (LN) depends on its sophisticated tissue architecture, consisting of several subcompartments supported by distinct fibroblastic stromal cells (FSCs). However, the intricate details of stromal structures and associated FSC subsets are not fully understood. Using several gene reporter mice, we sought to discover unrecognized stromal structures and FSCs in the LN. The four previously identified FSC subsets in the cortex are clearly distinguished by the expression pattern of reporters including PDGFRß, CCL21-ser, and CXCL12. Herein, we identified a unique FSC subset expressing both CCL21-ser and CXCL12 in the deep cortex periphery (DCP) that is characterized by preferential B cell localization. This subset was clearly different from CXCL12highLepRhigh FSCs in the medullary cord, which harbors plasma cells. B cell localization in the DCP was controlled chiefly by CCL21-ser and, to a lesser extent, CXCL12. Moreover, the optimal development of the DCP as well as medulla requires B cells. Together, our findings suggest the presence of a unique microenvironment in the cortex-medulla boundary and offer an advanced view of the multi-layered stromal framework constructed by distinct FSC subsets in the LN.

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.

Extensively re-organized systemic lymph nodes provide a feasible environment for self-reactivity in lupus-prone NZB × NZW F1 mice.

Lymphadenopathy is a frequently observed symptom in systemic lupus erythematosus, although the immunological role of lymph nodes (LNs) in systemic autoimmunity remains largely unknown. Here, we performed comprehensive and systematic analyses of LNs in lupus-prone NZB × NZW F1 (BWF1) mice, demonstrating extensive tissue re-organization of the systemic LNs with follicular expansion, hyper germinal center (GC) formation, atrophy of the paracortical T-cell area and expansion of the medulla in aged BWF1 mice bearing glomerulonephritis. The proportion of B cells was significantly increased in these reactive LNs but not in the spleen, and lymphocyte subsets involved in antibody production, i.e. GC B cells, follicular helper T cells and plasma cells, were elevated. Draining LNs of the affected organs, such as the renal and cervical nodes, showed enhanced tissue re-organization and accumulation of effector lymphocytes, suggesting the presence of a positive feedback loop of regional responses. LN cells isolated from disease-bearing animals produced anti-DNA antibody, indicating activation of autoreactive lymphocytes in situ. The substantial development of disease and LN alterations in mice that received a splenectomy at a young age points to the importance of other secondary lymphoid organs, most likely LNs, for the progression of autoimmune responses independent of the spleen. Taken together, our findings highlight the value of taking LN alterations and activities into consideration for understanding the pathogenesis of systemic autoimmunity.

CD4 CTL, a Cytotoxic Subset of CD4+ T Cells, Their Differentiation and Function.

CD4+ T cells with cytotoxic activity (CD4 CTL) have been observed in various immune responses. These cells are characterized by their ability to secrete granzyme B and perforin and to kill the target cells in an MHC class II-restricted fashion. Although CD4 CTLs were once thought to be an in vitro artifact associated with long-term culturing, they have since been identified in vivo and shown to play important roles in antiviral and antitumor immunity, as well as in inflammation. Functional characterization of CD4 CTL suggests their potential significance for therapeutic purposes. However, in order to develop effective CD4 CTL therapy it is necessary to understand the differentiation and generation of these cells. Although the mechanisms regulating development of various CD4+ Th subsets have been clarified in terms of the cytokine and transcription factor requirement, the CD4 CTL differentiation mechanism remains elusive. These cells are thought to be most closely related to Th1 cells secreting IFNγ and regulated by eomesodermin and/or T-bet transcription factors for their differentiation. However, our studies and those of others have identified CD4 CTLs within other CD4+ T cell subsets, including naïve T cells. We have identified class I-restricted T cell-associated molecule as a marker of CD4 CTL and, by using this marker, we detected a subset of naïve T cells that have the potential to differentiate into CD4 CTL. CD4 CTL develops at sites of infections as well as inflammation. In this review, we summarize recent findings about the generation of CD4 CTL and propose a model with several differentiation pathways.

CRTAM determines the CD4+ cytotoxic T lymphocyte lineage.

Naive T cells differentiate into various effector T cells, including CD4(+) helper T cell subsets and CD8(+) cytotoxic T cells (CTL). Although cytotoxic CD4(+) T cells (CD4 +: CTL) also develop from naive T cells, the mechanism of development is elusive. We found that a small fraction of CD4(+) T cells that express class I-restricted T cell-associated molecule (CRTAM) upon activation possesses the characteristics of both CD4(+) and CD8(+) T cells. CRTAM(+) CD4(+) T cells secrete IFN-γ, express CTL-related genes, such as eomesodermin (Eomes), Granzyme B, and perforin, after cultivation, and exhibit cytotoxic function, suggesting that CRTAM(+) T cells are the precursor of CD4(+)CTL. Indeed, ectopic expression of CRTAM in T cells induced the production of IFN-γ, expression of CTL-related genes, and cytotoxic activity. The induction of CD4(+)CTL and IFN-γ production requires CRTAM-mediated intracellular signaling. CRTAM(+) T cells traffic to mucosal tissues and inflammatory sites and developed into CD4(+)CTL, which are involved in mediating protection against infection as well as inducing inflammatory response, depending on the circumstances, through IFN-γ secretion and cytotoxic activity. These results reveal that CRTAM is critical to instruct the differentiation of CD4(+)CTL through the induction of Eomes and CTL-related gene.

Bach2 maintains T cells in a naive state by suppressing effector memory-related genes.

The transcriptional repressor BTB and CNC homology 2 (Bach2) is thought to be mainly expressed in B cells with specific functions such as class switch recombination and somatic hypermutation, but its function in T cells is not known. We found equal Bach2 expression in T cells and analyzed its function using Bach2-deficient (-/-) mice. Although T-cell development was normal, numbers of peripheral naive T cells were decreased, which rapidly produced Th2 cytokines after TCR stimulation. Bach2(-/-) naive T cells highly expressed genes related to effector-memory T cells such as CCR4, ST-2 and Blimp-1. Enhanced expression of these genes induced Bach2(-/-) naive T cells to migrate toward CCR4-ligand and respond to IL33. Forced expression of Bach2 restored the expression of these genes. Using Chromatin Immunoprecipitation (ChIP)-seq analysis, we identified S100 calcium binding protein a, Heme oxigenase 1, and prolyl hydroxylase 3 as Bach2 direct target genes, which are highly expressed in effector-memory T cells. These findings indicate that Bach2 suppresses effector memory-related genes to maintain the naive T-cell state and regulates generation of effector-memory T cells.

CRTAM confers late-stage activation of CD8+ T cells to regulate retention within lymph node.

In vivo immune response is triggered in the lymph node, where lymphocytes for entry into, retention at, and migration to effector sites are dynamically regulated. The molecular mechanism underlying retention regulation is the key to elucidating in vivo regulation of immune response. In this study, we describe the function of the adhesion molecule class I-restricted T cell-associated molecule (CRTAM) in regulating CD8+ T cell retention within the lymph node and eventually effector function. We previously identified CRTAM as a receptor predominantly expressed on activated CD8+ T cells, and nectin-like molecule-2 (Necl2) as its ligand. In vivo function of CRTAM-Necl2 interaction was analyzed by generating CRTAM(-/-) mice. CRTAM(-/-) mice exhibited reduced protective immunity against viral infection and impaired autoimmune diabetes induction in vivo. Although Ag-specific CRTAM(-/-) CD8+ T cells showed normal CTL functions in vitro, their number in the draining lymph node was reduced. Because CRTAM+ T cells bound efficiently to Necl2-expressing CD8+ dendritic cells (DCs) that reside in T cell area of lymph node, CRTAM may induce retention by binding to CD8+ DCs at the late stage of activation before proliferation. The CRTAM-mediated late interaction with DCs induced retention of activated CD8+ T cells in an Ag-independent fashion, and this possibly resulted in effective CTL development in the draining lymph node.

Cell type-specific regulation of ITAM-mediated NF-kappaB activation by the adaptors, CARMA1 and CARD9.

Activating NK cell receptors transduce signals through ITAM-containing adaptors, including FcRgamma and DAP12. Although the caspase recruitment domain (CARD)9-Bcl10 complex is essential for FcRgamma/DAP12-mediated NF-kappaB activation in myeloid cells, its involvement in NK cell receptor signaling is unknown. Herein we show that the deficiency of CARMA1 or Bcl10, but not CARD9, resulted in severe impairment of cytokine/chemokine production mediated by activating NK cell receptors due to a selective defect in NF-kappaB activation, whereas cytotoxicity mediated by the same receptors did not require CARMA1-Bcl10-mediated signaling. IkappaB kinase (IKK) activation by direct protein kinase C (PKC) stimulation with PMA plus ionomycin (P/I) was abrogated in CARMA1-deficient NK cells, similar to T and B lymphocytes, whereas CARD9-deficient dendritic cells (DCs) exhibited normal P/I-induced IKK activation. Surprisingly, CARMA1 deficiency also abrogated P/I-induced IKK activation in DCs, indicating that CARMA1 is essential for PKC-mediated NF-kappaB activation in all cell types, although the PKC-CARMA1 axis is not used downstream of myeloid ITAM receptors. Consistently, PKC inhibition abrogated ITAM receptor-mediated activation only in NK cells but not in DCs, suggesting PKC-CARMA1-independent, CARD9-dependent ITAM receptor signaling in myeloid cells. Conversely, the overexpression of CARD9 in CARMA1-deficient cells failed to restore the PKC-mediated NF-kappaB activation. Thus, NF-kappaB activation signaling through ITAM receptors is regulated by a cell type-specific mechanism depending on the usage of adaptors CARMA1 and CARD9, which determines the PKC dependence of the signaling.

Cloning of B cell-specific membrane tetraspanning molecule BTS possessing B cell proliferation-inhibitory function.

Lymphocyte proliferation is regulated by signals through antigen receptors, co-stimulatory receptors, and other positive and negative modulators. Several membrane tetraspanning molecules are also involved in the regulation of lymphocyte growth and death. We cloned a new B cell-specific tetraspanning (BTS) membrane molecule, which is similar to CD20 in terms of expression, structure and function. BTS is specifically expressed in the B cell line and its expression is increased after the pre-B cell stage. BTS is expressed in intracellular granules and on the cell surface. Overexpression of BTS in immature B cell lines induces growth retardation through inhibition of cell cycle progression and cell size increase without inducing apoptosis. This inhibitory function is mediated predominantly by the N terminus of BTS. The development of mature B cells is inhibited in transgenic mice expressing BTS, suggesting that BTS is involved in the in vivo regulation of B cells. These results indicate that BTS plays a role in the regulation of cell division and B cell growth.

The adaptor protein CARD9 is essential for the activation of myeloid cells through ITAM-associated and Toll-like receptors.

Immunoreceptor tyrosine-based activation motifs (ITAMs) are crucial in antigen receptor signaling in acquired immunity. Although receptors associated with the ITAM-bearing adaptors FcRgamma and DAP12 on myeloid cells have been suggested to activate innate immune responses, the mechanism coupling those receptors to 'downstream' signaling events is unclear. The CARMA1-Bcl-10-MALT1 complex is critical for the activation of transcription factor NF-kappaB in lymphocytes but has an unclear function in myeloid cells. Here we report that deletion of the gene encoding the Bcl-10 adaptor-binding partner CARD9 resulted in impaired myeloid cell activation of NF-kappaB signaling by several ITAM-associated receptors. Moreover, CARD9 was required for Toll-like receptor-induced activation of dendritic cells through the activation of mitogen-activated protein kinases. Although Bcl10-/- and Card9-/- mice had similar signaling impairment in myeloid cells, Card11-/- (CARMA1-deficient) myeloid cell responses were normal, and although Card11-/- lymphocytes were defective in antigen receptor-mediated activation, Card9-/- lymphocytes were not. Thus, the activation of lymphoid and myeloid cells through ITAM-associated receptors or Toll-like receptors is regulated by CARMA1-Bcl-10 and CARD9-Bcl-10, respectively.

A critical role for the innate immune signaling molecule IRAK-4 in T cell activation.

IRAK-4 is a protein kinase that is pivotal in mediating signals for innate immune responses. Here, we report that IRAK-4 signaling is also essential for eliciting adaptive immune responses. Thus, in the absence of IRAK-4, in vivo T cell responses were significantly impaired. Upon T cell receptor stimulation, IRAK-4 is recruited to T cell lipid rafts, where it induces downstream signals, including protein kinase C activation through the association with Zap70. This signaling pathway was found to be required for optimal activation of nuclear factor kappaB. Our findings suggest that T cells use this critical regulator of innate immunity for the development of acquired immunity, suggesting that IRAK-4 may be involved in direct signal cross talk between the two systems.

DNA polymerase theta contributes to the generation of C/G mutations during somatic hypermutation of Ig genes.

Somatic hypermutation of Ig variable region genes is initiated by activation-induced cytidine deaminase; however, the activity of multiple DNA polymerases is required to ultimately introduce mutations. DNA polymerase eta (Poleta) has been implicated in mutations at A/T, but polymerases involved in C/G mutations have not been identified. We have generated mutant mice expressing DNA polymerase (Pol) specifically devoid of polymerase activity. Compared with WT mice, Polq-inactive (Polq, the gene encoding Pol) mice exhibited a reduced level of serum IgM and IgG1. The mutant mice mounted relatively normal primary and secondary immune responses to a T-dependent antigen, but the production of high-affinity specific antibodies was partially impaired. Analysis of the J(H)4 intronic sequences revealed a slight reduction in the overall mutation frequency in Polq-inactive mice. Remarkably, although mutations at A/T were unaffected, mutations at C/G were significantly decreased, indicating an important, albeit not exclusive, role for Pol activity. The reduction of C/G mutations was particularly focused on the intrinsic somatic hypermutation hotspots and both transitions and transversions were similarly reduced. These findings, together with the recent observation that Pol efficiently catalyzes the bypass of abasic sites, lead us to propose that Pol introduces mutations at C/G by replicating over abasic sites generated via uracil-DNA glycosylase.

Heterotypic interaction of CRTAM with Necl2 induces cell adhesion on activated NK cells and CD8+ T cells.

NK cells and CD8+ T cells exhibit cytotoxicity and cytokine production upon recognizing target cells through cell-cell interaction. We screened the molecules involved in the recognition and regulation of these cells using cDNA subtraction between naive and activated NK cells. We identified class I-restricted T cell-associated molecule (CRTAM), a two Ig domain-bearing surface receptor, as a molecule rapidly and transiently expressed on NK cells and CD8+ T cells upon activation. CRTAM is expressed as a dimer on the cell surface, and its expression is transcriptionally regulated. Using an expression-cloning system, we then further identified Nectin-like (Necl) molecule 2, a three Ig domain-containing receptor, as a ligand of CRTAM. While Necl2 mediates homotypic interaction, CRTAM interacts with Necl2 but not with CRTAM itself. The heterotypic CRTAM-Necl2 interaction has a higher affinity than the homotypic Necl2 interaction. Although there was no clear alteration in the cytotoxic function of the NK cells and CD8+ T cells against the Necl2-expressing target cells, T cells expressing CRTAM tightly bound to Necl2-expressing cells. CRTAM+ cells did not induce homotypic aggregation but they did exert strong heterotypic binding with Necl2+ cells, which was inhibited by the addition of the CRTAM-Ig fusion protein. These results suggest that the heterotypic interaction between CRTAM and Necl2 plays an important role in the adhesion, interaction or migration of NK cells and CD8+ T cells upon stimulation.