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.

Development of organ-specific autoimmunity by dysregulated Aire expression.

Deficiency for AIRE/Aire in both humans and mice results in the development of organ-specific autoimmune disease. We tested whether augmented and/or dysregulated AIRE/Aire expression might be also prone to the breakdown of self-tolerance. To define the effect of augmented Aire expression on the development of autoimmunity, antigen-specific clonal deletion and production of clonotypic regulatory T cells (Tregs) in the thymus were examined using mice expressing two additional copies of Aire in a heterozygous state (3xAire-knockin mice: 3xAire-KI). We found that both clonal deletion of autoreactive T cells and production of clonotypic Tregs in the thymus from 3xAire-KI were impaired in a T-cell receptor-transgenic system. Furthermore, 3xAire-KI females showed higher scores of experimental autoimmune encephalomyelitis induced by myelin oligodendrocyte glycoprotein than wild-type littermates, suggesting that augmented Aire expression exacerbates organ-specific autoimmunity under disease-prone conditions. In humans, we found that one patient with amyopathic dermatomyositis showed CD3- CD19- cells expressing AIRE in the peripheral blood before the treatment but not during the remission phase treated with immunosuppressive drugs. Thus, not only loss of function of AIRE/Aire but also augmented and/or dysregulated expression of AIRE/Aire should be considered for the pathogenesis of organ-specific autoimmunity. We suggest that further analyses should be pursued to establish a novel link between organ-specific autoimmune disease and dysregulated AIRE expression in clinical settings.

Aire Controls Heterogeneity of Medullary Thymic Epithelial Cells for the Expression of Self-Antigens.

The deficiency of Aire, a transcriptional regulator whose defect results in the development of autoimmunity, is associated with reduced expression of tissue-restricted self-Ags (TRAs) in medullary thymic epithelial cells (mTECs). Although the mechanisms underlying Aire-dependent expression of TRAs need to be explored, the physical identification of the target(s) of Aire has been hampered by the low and promiscuous expression of TRAs. We have tackled this issue by engineering mice with augmented Aire expression. Integration of the transcriptomic data from Aire-augmented and Aire-deficient mTECs revealed that a large proportion of so-called Aire-dependent genes, including those of TRAs, may not be direct transcriptional targets downstream of Aire. Rather, Aire induces TRA expression indirectly through controlling the heterogeneity of mTECs, as revealed by single-cell analyses. In contrast, Ccl25 emerged as a canonical target of Aire, and we verified this both in vitro and in vivo. Our approach has illuminated the Aire's primary targets while distinguishing them from the secondary targets.

Tissue-specific autoimmunity controlled by Aire in thymic and peripheral tolerance mechanisms.

Tissue-specific autoimmune diseases are assumed to arise through malfunction of two checkpoints for immune tolerance: defective elimination of autoreactive T cells in the thymus and activation of these T cells by corresponding autoantigens in the periphery. However, evidence for this model and the outcome of such alterations in each or both of the tolerance mechanisms have not been sufficiently investigated. We studied these issues by expressing human AIRE (huAIRE) as a modifier of tolerance function in NOD mice wherein the defects of thymic and peripheral tolerance together cause type I diabetes (T1D). Additive huAIRE expression in the thymic stroma had no major impact on the production of diabetogenic T cells in the thymus. In contrast, huAIRE expression in peripheral antigen-presenting cells (APCs) rendered the mice resistant to T1D, while maintaining other tissue-specific autoimmune responses and antibody production against an exogenous protein antigen, because of the loss of Xcr1+ dendritic cells, an essential component for activating diabetogenic T cells in the periphery. These results contrast with our recent demonstration that huAIRE expression in both the thymic stroma and peripheral APCs resulted in the paradoxical development of muscle-specific autoimmunity. Our results reveal that tissue-specific autoimmunity is differentially controlled by a combination of thymic function and peripheral tolerance, which can be manipulated by expression of huAIRE/Aire in each or both of the tolerance mechanisms.

Aire Controls in Trans the Production of Medullary Thymic Epithelial Cells Expressing Ly-6C/Ly-6G.

Medullary thymic epithelial cells (mTECs), which express a wide range of tissue-restricted Ags (TRAs), contribute to the establishment of self-tolerance by eliminating autoreactive T cells and/or inducing regulatory T cells. Aire controls a diverse set of TRAs within Aire-expressing cells by employing various transcriptional pathways. As Aire has a profound effect on transcriptomes of mTECs, including TRAs not only at the single-cell but also the population level, we suspected that Aire (Aire+ mTECs) might control the cellular composition of the thymic microenvironment. In this study, we confirmed that this is indeed the case by identifying a novel mTEC subset expressing Ly-6 family protein whose production was defective in Aire-deficient thymi. Reaggregated thymic organ culture experiments demonstrated that Aire did not induce the expression of Ly-6C/Ly-6G molecules from mTECs as Aire-dependent TRAs in a cell-intrinsic manner. Instead, Aire+ mTECs functioned in trans to maintain Ly-6C/Ly-6G+ mTECs. Thus, Aire not only controls TRA expression transcriptionally within the cell but also controls the overall composition of mTECs in a cell-extrinsic manner, thereby regulating the transcriptome from mTECs on a global scale.

Paradoxical development of polymyositis-like autoimmunity through augmented expression of autoimmune regulator (AIRE).

Autoimmunity is prevented by the function of the autoimmune regulator [AIRE (Aire in mice)], which promotes the expression of a wide variety of tissue-restricted antigens (TRAs) from medullary thymic epithelial cells (mTECs) and from a subset of peripheral antigen-presenting cells (APCs). We examined the effect of additive expression of human AIRE (huAIRE) in a model of autoimmune diabetes in NOD mice. Unexpectedly, we observed that mice expressing augmented AIRE/Aire developed muscle-specific autoimmunity associated with incomplete maturation of mTECs together with impaired expression of Aire-dependent TRAs. This led to failure of deletion of autoreactive T cells together with dramatically reduced production of regulatory T cells in the thymus. In peripheral APCs, expression of costimulatory molecules was augmented. We suggest that levels of Aire expression need to be tightly controlled for maintenance of immunological tolerance. Our results also highlight the importance of coordinated action between central tolerance and peripheral tolerance under the common control of Aire.

Actin Family Proteins in the Human INO80 Chromatin Remodeling Complex Exhibit Functional Roles in the Induction of Heme Oxygenase-1 with Hemin.

Nuclear actin family proteins, comprising of actin and actin-related proteins (Arps), are essential functional components of the multiple chromatin remodeling complexes. The INO80 chromatin remodeling complex, which is evolutionarily conserved and has roles in transcription, DNA replication and repair, consists of actin and actin-related proteins Arp4, Arp5, and Arp8. We generated Arp5 knockout (KO) and Arp8 KO cells from the human Nalm-6 pre-B cell line and used these KO cells to examine the roles of Arp5 and Arp8 in the transcriptional regulation mediated by the INO80 complex. In both of Arp5 KO and Arp8 KO cells, the oxidative stress-induced expression of HMOX1 gene, encoding for heme oxygenase-1 (HO-1), was significantly impaired. Consistent with these observations, chromatin immunoprecipitation (ChIP) assay revealed that oxidative stress caused an increase in the binding of the INO80 complex to the regulatory sites of HMOX1 in wild-type cells. The binding of INO80 complex to chromatin was reduced in Arp8 KO cells compared to that in the wild-type cells. On the other hand, the binding of INO80 complex to chromatin in Arp5 KO cells was similar to that in the wild-type cells even under the oxidative stress condition. However, both remodeling of chromatin at the HMOX1 regulatory sites and binding of a transcriptional activator to these sites were impaired in Arp5 KO cells, indicating that Arp5 is required for the activation of the INO80 complex. Collectively, these results suggested that these nuclear Arps play indispensable roles in the function of the INO80 chromatin remodeling complex.

Aire Expression Is Inherent to Most Medullary Thymic Epithelial Cells during Their Differentiation Program.

Aire in medullary thymic epithelial cells (mTECs) plays an important role in the establishment of self-tolerance. Because Aire(+) mTECs appear to be a limited subset, they may constitute a unique lineage(s) among mTECs. An alternative possibility is that all mTECs are committed to express Aire in principle, but Aire expression by individual mTECs is conditional. To investigate this issue, we established a novel Aire reporter strain in which endogenous Aire is replaced by the human AIRE-GFP-Flag tag (Aire/hAGF-knockin) fusion gene. The hAGF reporter protein was produced and retained very efficiently within mTECs as authentic Aire nuclear dot protein. Remarkably, snapshot analysis revealed that mTECs expressing hAGF accounted for >95% of mature mTECs, suggesting that Aire expression does not represent a particular mTEC lineage(s). We confirmed this by generating Aire/diphtheria toxin receptor-knockin mice in which long-term ablation of Aire(+) mTECs by diphtheria toxin treatment resulted in the loss of most mature mTECs beyond the proportion of those apparently expressing Aire. These results suggest that Aire expression is inherent to all mTECs but may occur at particular stage(s) and/or cellular states during their differentiation, thus accounting for the broad impact of Aire on the promiscuous gene expression of mTECs.

Ectopic Aire Expression in the Thymic Cortex Reveals Inherent Properties of Aire as a Tolerogenic Factor within the Medulla.

Cortical thymic epithelial cells (cTECs) and medullary thymic epithelial cells (mTECs) play essential roles in the positive and negative selection of developing thymocytes, respectively. Aire in mTECs plays an essential role in the latter process through expression of broad arrays of tissue-restricted Ags. To determine whether the location of Aire within the medulla is absolutely essential or whether Aire could also function within the cortex for establishment of self-tolerance, we used bacterial artificial chromosome technology to establish a semiknockin strain of NOD-background (ß5t/Aire-transgenic) mice expressing Aire under control of the promoter of ß5t, a thymoproteasome expressed exclusively in the cortex. Although Aire was expressed in cTECs as typical nuclear dot protein in ß5t/Aire-Tg mice, cTECs expressing Aire ectopically did not confer transcriptional expression of either Aire-dependent or Aire-independent tissue-restricted Ag genes. We then crossed ß5t/Aire-Tg mice with Aire-deficient NOD mice, generating a strain in which Aire expression was confined to cTECs. Despite the presence of Aire(+) cTECs, these mice succumbed to autoimmunity, as did Aire-deficient NOD mice. The thymic microenvironment harboring Aire(+) cTECs, within which many Aire-activated genes were present, also showed no obvious alteration of positive selection, suggesting that Aire's unique property of generating a self-tolerant T cell repertoire is functional only in mTECs.

DNA binding properties of the actin-related protein Arp8 and its role in DNA repair.

Actin and actin-related proteins (Arps), which are members of the actin family, are essential components of many of these remodeling complexes. Actin, Arp4, Arp5, and Arp8 are found to be evolutionarily conserved components of the INO80 chromatin remodeling complex, which is involved in transcriptional regulation, DNA replication, and DNA repair. A recent report showed that Arp8 forms a module in the INO80 complex and this module can directly capture a nucleosome. In the present study, we showed that recombinant human Arp8 binds to DNAs, and preferentially binds to single-stranded DNA. Analysis of the binding of adenine nucleotides to Arp8 mutants suggested that the ATP-binding pocket, located in the evolutionarily conserved actin fold, plays a regulatory role in the binding of Arp8 to DNA. To determine the cellular function of Arp8, we derived tetracycline-inducible Arp8 knockout cells from a cultured human cell line. Analysis of results obtained after treating these cells with aphidicolin and camptothecin revealed that Arp8 is involved in DNA repair. Together with the previous observation that Arp8, but not γ-H2AX, is indispensable for recruiting INO80 complex to DSB in human, results of our study suggest an individual role for Arp8 in DNA repair.

NF-κB-inducing kinase in thymic stroma establishes central tolerance by orchestrating cross-talk with not only thymocytes but also dendritic cells.

Essential roles of NF-κB-inducing kinase (NIK) for the development of medullary thymic epithelial cells (mTECs) and regulatory T cells have been highlighted by studies using a strain of mouse bearing a natural mutation of the NIK gene (aly mice). However, the exact mechanisms underlying the defect in thymic cross-talk leading to the breakdown of self-tolerance in aly mice remain elusive. In this study, we demonstrated that production of regulatory T cells and the final maturation process of positively selected conventional αß T cells are impaired in aly mice, partly because of a lack of mature mTECs. Of note, numbers of thymic dendritic cells and their expression of costimulatory molecules were also affected in aly mice in a thymic stroma-dependent manner. The results suggest a pivotal role of NIK in the thymic stroma in establishing self-tolerance by orchestrating cross-talk between mTECs and dendritic cells as well as thymocytes. In addition, we showed that negative selection was impaired in aly mice as a result of the stromal defect, which accounts for the development of organ-specific autoimmunity through a lack of normal NIK.

Temporal lineage tracing of Aire-expressing cells reveals a requirement for Aire in their maturation program.

Understanding the cellular dynamics of Aire-expressing lineage(s) among medullary thymic epithelial cells (AEL-mTECs) is essential for gaining insight into the roles of Aire in establishment of self-tolerance. In this study, we monitored the maturation program of AEL-mTECs by temporal lineage tracing, in which bacterial artificial chromosome transgenic mice expressing tamoxifen-inducible Cre recombinase under control of the Aire regulatory element were crossed with reporter strains. We estimated that the half-life of AEL-mTECs subsequent to Aire expression was ∼7-8 d, which was much longer than that reported previously, owing to the existence of a post-Aire stage. We found that loss of Aire did not alter the overall lifespan of AEL-mTECs, inconsistent with the previous notion that Aire expression in medullary thymic epithelial cells (mTECs) might result in their apoptosis for efficient cross-presentation of self-antigens expressed by AEL-mTECs. In contrast, Aire was required for the full maturation program of AEL-mTECs, as exemplified by the lack of physiological downregulation of CD80 during the post-Aire stage in Aire-deficient mice, thus accounting for the abnormally increased CD80(high) mTECs seen in such mice. Of interest, increased CD80(high) mTECs in Aire-deficient mice were not mTEC autonomous and were dependent on cross-talk with thymocytes. These results further support the roles of Aire in the differentiation program of AEL-mTECs.

Which model better fits the role of aire in the establishment of self-tolerance: the transcription model or the maturation model?

The discovery of Aire-dependent transcriptional control of many tissue-restricted self-antigen (TRA) genes in thymic epithelial cells in the medulla (medullary thymic epithelial cells, mTECs) has raised the intriguing question of how the single Aire gene can influence the transcription of such a large number of TRA genes within mTECs. From a mechanistic viewpoint, there are two possible models to explain the function of Aire in this action. In the first model, TRAs are considered to be the direct target genes of Aire's transcriptional activity. In this scenario, the lack of Aire protein within cells would result in the defective TRA gene expression, while the maturation program of mTECs would be unaffected in principle. The second model hypothesizes that Aire is necessary for the maturation program of mTECs. In this case, we assume that the mTEC compartment does not mature normally in the absence of Aire. If acquisition of the properties of TRA gene expression depends on the maturation status of mTECs, a defect of such an Aire-dependent maturation program in Aire-deficient mTECs can also result in impaired TRA gene expression. In this brief review, we will focus on these two contrasting models for the roles of Aire in controlling the expression of TRAs within mTECs.

The integrator complex is required for integrity of Cajal bodies.

The nucleus in eukaryotic cells is a highly organized and dynamic structure containing numerous subnuclear bodies. The morphological appearance of nuclear bodies seems to be a reflection of ongoing functions, such as DNA replication, transcription, repair, RNA processing and RNA transport. The integrator complex mediates processing of small nuclear RNA (snRNA), so it might play a role in nuclear body formation. Here, we show that the integrator complex is essential for integrity of the Cajal body. Depletion of INTS4, an integrator complex subunit, abrogated 3'-end processing of snRNA. A defect in this activity caused a significant accumulation of the Cajal body marker protein coilin in nucleoli. Some fractions of coilin still formed nucleoplasmic foci; however, they were free of other Cajal body components, such as survival of motor neuron protein (SMN), Sm proteins and snRNAs. SMN and Sm proteins formed striking cytoplasmic granules. These findings demonstrate that the integrator complex is essential for snRNA maturation and Cajal body homeostasis.

Proteome analysis of human nuclear insoluble fractions.

The interphase nucleus is a highly ordered and compartmentalized organelle. Little is known regarding what elaborate mechanisms might exist to explain these properties of the nucleus. Also unresolved is whether some architectural components might facilitate the formation of functional intranuclear compartments or higher order chromatin structure. As the first step to address these questions, we performed an in-depth proteome analysis of nuclear insoluble fractions of human HeLa-S3 cells prepared by two different approaches: a high-salt/detergent/nuclease-resistant fraction and a lithium 3,5-diiodosalicylate/nuclease-resistant fraction. Proteins of the fractions were analyzed by liquid chromatography electrospray ionization tandem mass spectrometry, identifying 333 and 330 proteins from each fraction respectively. Among the insoluble nuclear proteins, we identified 37 hitherto unknown or functionally uncharacterized proteins. The RNA recognition motif, WD40 repeats, HEAT repeats and the SAP domain were often found in these identified proteins. The subcellular distribution of selected proteins, including DEK protein and SON protein, demonstrated their novel associations with nuclear insoluble materials, corroborating our MS-based analysis. This study establishes a comprehensive catalog of the nuclear insoluble proteins in human cells. Further functional analysis of the proteins identified in our study will significantly improve our understanding of the dynamic organization of the interphase nucleus.

Generation of tetracycline-inducible conditional gene knockout cells in a human Nalm-6 cell line.

Conditional gene knockout by homologous recombination combined with an inducible gene expression system is a powerful approach for studying gene function, although homologous recombination in human cells occurs infrequently. The tetracycline-regulated gene expression (Tet-Off) system is a convenient method for achieving conditional gene knockout, but it is not always promising in Nalm-6, a rare human cell line highly effective for gene targeting. Here we modified the Tet-Off system and applied it to the Nalm-6 cell line successfully by using an internal ribosome entry site to drive a selectable marker from the same tetracycline-responsive promoter for the transgene. We also inserted the gene for the tetracycline-controlled transactivator under the control of a potent CAG promoter. These modifications enabled us to easily obtain rare clones that express optimal amounts of tetracycline-regulated transgenes. We thereby generated a 'tetracycline-inducible conditional gene knockout' for the proliferation-associated SNF2-like gene (PASG) in a Nalm-6 cell line, in which the expression of PASG can be depleted in a tetracycline-dependent manner on a knockout background. This method is applicable to any human genes, making this gene-targeting system using the Nalm-6 cell line a promising tool for analyzing gene function.

Improved applications of the tetracycline-regulated gene depletion system.

Tightly controlled expression of transgenes in mammalian cells is an important tool for biological research, drug discovery, and future genetic therapies. The tetracycline-regulated gene depletion (Tet-Off) system has been widely used to control gene activities in mammalian cells, because it allows strict regulation of transgenes but no pleiotropic effects of prokaryotic regulatory proteins. However, the Tet-Off system is not compatible with every cell type and this is the main remaining obstacle left for this system. Recently, we overcame this problem by inserting an internal ribosome entry site (IRES) to drive a selectable marker from the same tetracycline-responsive promoter for the transgene. We also employed a CMV immediate early enhancer/beta-actin (CAG) promoter to express a Tet-controlled transactivator. Indeed, the Tet-Off system with these technical modifications was applied successfully to the human pre-B Nalm-6 cell line in which conventional Tet-Off systems had not worked efficiently. These methodological improvements should be applicable for many other mammalian proliferating cells. In this review we give an overview and introduce a new method for the improved application of the Tet-Off system.

Histone acetyltransferase-1 regulates integrity of cytosolic histone H3-H4 containing complex.

Amounts of soluble histones in cells are tightly regulated to ensure supplying them for the newly synthesized DNA and preventing the toxic effect of excess histones. Prior to incorporation into chromatin, newly synthesized histones H3 and H4 are highly acetylated in pre-deposition complex, wherein H4 is di-acetylated at Lys-5 and Lys-12 residues by histone acetyltransferase-1 (Hat1), but their role in histone metabolism is still unclear. Here, using chicken DT 40 cytosolic extracts, we found that histones H3/H4 and their chaperone Asf1, including RbAp48, a regulatory subunit of Hat1 enzyme, were associated with Hat1. Interestingly, in HAT1-deficient cells, cytosolic histones H3/H4 fractions on sucrose gradient centrifugation, having a sedimentation coefficient of 5-6S in DT40 cells, were shifted to lower molecular mass fractions, with Asf1. Further, sucrose gradient fractionation of semi-purified tagged Asf1-complexes showed the presence of Hat1, RbAp48 and histones H3/H4 at 5-6S fractions in the complexes. These findings suggest the possible involvement of Hat1 in regulating cytosolic H3/H4 pool mediated by Asf1-containing cytosolic H3/H4 pre-deposition complex.

Schizosaccharomyces pombe Snf2SR, a novel SNF2 family protein, interacts with Ran GTPase and modulates both RanGEF and RanGAP activities.

Snf2SR, a suppressor of rna1(ts), which is a temperature-sensitive mutation in Schizosaccharomyces pombe RanGAP (GTPase activating protein), possesses both the SNF2 and the helicase domains conserved in the chromatin remodeling SNF2 ATPase/helicase protein family. We have now clarified a function of Snf2SR. Snf2SR indeed showed DNA-stimulated ATPase activity, proving that it is a member of the SNF2 ATPase/helicase family. Consistent with this role, Snf2SR was localized in the nucleus and cell fractionation analysis revealed that Snf2SR was tightly associated with the nuclear matrix. The disruption of snf2SR(+) was detrimental for a cell proliferation of S. pombe. Snf2SR that did not enhance RanGAP activity by itself, but abolished histone-H3-mediated RanGAP inhibition, as previously reported for the histone H3 methyltransferase, Clr4, another rna1(ts) suppressor. In contrast to Clr4, Snf2SR directly bound to the GDP-bound form of the S. pombe Ran homologue Spi1 and enhanced the nucleotide exchange activity of Pim1, the S. pombe RanGEF (guanine nucleotide exchange factor). Over-expression of Spi1-G18V, a Ran GTPase mutant fixed in the GTP-bound form, was lethal to S. pombe Deltasnf2SR. Together, our results indicate that Snf2SR is involved in the Ran GTPase cycle in vivo.