Novel anti-GARP antibody DS-1055a augments anti-tumor immunity by depleting highly suppressive GARP+ regulatory T cells.

Regulatory T (Treg) cells, which are essential for maintaining self-tolerance, inhibit anti-tumor immunity, consequently hindering protective cancer immunosurveillance, and hampering effective anti-tumor immune responses in tumor-bearing hosts. Here, we show that depletion of Treg cells via targeting glycoprotein A repetitions predominant (GARP) induces effective anti-tumor immune responses. GARP was specifically expressed by highly suppressive Treg cells in the tumor microenvironment (TME) of multiple cancer types in humans. In the periphery, GARP was selectively induced in Treg cells, but not in effector T cells, by polyclonal stimulation. DS-1055a, a novel afucosylated anti-human GARP monoclonal antibody, efficiently depleted GARP+ Treg cells, leading to the activation of effector T cells. Moreover, DS-1055a decreased FoxP3+CD4+ T cells in the TME and exhibited remarkable anti-tumor activity in humanized mice bearing HT-29 tumors. We propose that DS-1055a is a new Treg-cell-targeted cancer immunotherapy agent with augmentation of anti-tumor immunity.

Dependence on nuclear factor of activated T-cells (NFAT) levels discriminates conventional T cells from Foxp3+ regulatory T cells.

Several lines of evidence suggest nuclear factor of activated T-cells (NFAT) to control regulatory T cells: thymus-derived naturally occurring regulatory T cells (nTreg) depend on calcium signals, the Foxp3 gene harbors several NFAT binding sites, and the Foxp3 (Fork head box P3) protein interacts with NFAT. Therefore, we investigated the impact of NFAT on Foxp3 expression. Indeed, the generation of peripherally induced Treg (iTreg) by TGF-ß was highly dependent on NFAT expression because the ability of CD4(+) T cells to differentiate into iTreg diminished markedly with the number of NFAT family members missing. It can be concluded that the expression of Foxp3 in TGF-ß-induced iTreg depends on the threshold value of NFAT rather than on an individual member present. This is specific for iTreg development, because frequency of nTreg remained unaltered in mice lacking NFAT1, NFAT2, or NFAT4 alone or in combination. Different from expectation, however, the function of both nTreg and iTreg was independent on robust NFAT levels, reflected by less nuclear NFAT in nTreg and iTreg. Accordingly, absence of one or two NFAT members did not alter suppressor activity in vitro or during colitis and transplantation in vivo. This scenario emphasizes an inhibition of high NFAT activity as treatment for autoimmune diseases and in transplantation, selectively targeting the proinflammatory conventional T cells, while keeping Treg functional.

Output feedback full-order sliding mode control for a three-tank system.

A robust output feedback nonlinear control method is proposed for output tracking of an uncertain three-tank system by using only the level sensor of the target tank. By using a coordinate transformation, we first transform the system model into a canonical form with uncertainties. The canonical system's state variables are estimated by a higher-order sliding mode differentiator based on the measurement of the target tank's liquid level. Then based on the estimated state variables, a continuous full-order sliding mode controller is constructed which can eliminate the effects of both the additive and multiplicative uncertainties. Rigorous analysis is carried out to clarify the control performance. And the effectiveness of the proposed method is verified by experimental studies on the Inteco Multi-tank system.

GARP as a Therapeutic Target for the Modulation of Regulatory T Cells in Cancer and Autoimmunity.

Regulatory T cells (Treg) play a critical role in immune homeostasis by suppressing several aspects of the immune response. Herein, Glycoprotein A repetitions predominant (GARP), the docking receptor for latent transforming growth factor (LTGF-ß), which promotes its activation, plays a crucial role in maintaining Treg mediated immune tolerance. After activation, Treg uniquely express GARP on their surfaces. Due to its location and function, GARP may represent an important target for immunotherapeutic approaches, including the inhibition of Treg suppression in cancer or the enhancement of suppression in autoimmunity. In the present review, we will clarify the cellular and molecular regulation of GARP expression not only in human Treg but also in other cells present in the tumor microenvironment. We will also examine the overall roles of GARP in the regulation of the immune system. Furthermore, we will explore potential applications of GARP as a predictive and therapeutic biomarker as well as the targeting of GARP itself in immunotherapeutic approaches.

A novel membrane protein, encoded by the gene covering KIAA0233, is transcriptionally induced in senile plaque-associated astrocytes.

Beta-amyloid (Abeta) deposition and senile plaque-associated astrocytes are common neuropathological features of Alzheimer's disease (AD). Although the molecular mechanisms by which Abeta contributes to the progression of neuropathologic changes have not been entirely established, there is little doubt that the association of Abeta with astrocytes, the predominant cell type in brain, significantly influences exacerbation of the disease. In an effort to identify astrocyte-derived molecules that may be intimately associated with progression of AD, we identified a novel Abeta-induced rat gene, designated Mib, whose human counterpart covers KIAA0233. Mib-transfected C6 cells express Mib protein in the endoplasmic reticulum and endplasmic reticulum-Golgi-intermediate compartment. To evaluate roles of Mib in AD, we investigated its expression in the AD brain. In non-AD brains, Mib mRNA has been detected in neurons but not in quiescent astrocytes. On the contrary, in AD brains, Mib mRNA is expressed in activated astrocytes associated with senile plaques, but not expressed in neurons around lesions. From these observations, Mib appears to be a novel Abeta-responsive gene that may play a role in astrocyte inflammatory activation around senile plaques in the AD brain.

Kinome Profiling of Regulatory T Cells: A Closer Look into a Complex Intracellular Network.

Regulatory T cells (Treg) are essential for T cell homeostasis and maintenance of peripheral tolerance. They prevent activation of auto-reactive T effector cells (Teff) in the context of autoimmunity and allergy. Otherwise, Treg also inhibit effective immune responses against tumors. Besides a number of Treg-associated molecules such as Foxp3, CTLA-4 or GARP, known to play critical roles in Treg differentiation, activation and function, the involvement of additional regulatory elements is suggested. Herein, kinase activities seem to play an important role in Treg fine tuning. Nevertheless, our knowledge regarding the complex intracellular signaling pathways controlling phenotype and function of Treg is still limited and based on single kinase cascades so far. To gain a more comprehensive insight into the pathways determining Treg function we performed kinome profiling using a phosphorylation-based kinome array in human Treg at different activation stages compared to Teff. Here we have determined intriguing quantitative differences in both populations. Resting and activated Treg showed an altered pattern of CD28-dependent kinases as well as of those involved in cell cycle progression. Additionally, significant up-regulation of distinct kinases such as EGFR or CK2 in activated Treg but not in Teff not only resemble data we obtained in previous studies in the murine system but also suggest that those specific molecular activation patterns can be used for definition of the activation and functional state of human Treg. Taken together, detailed investigation of kinome profiles opens the possibility to identify novel molecular mechanisms for a better understanding of Treg biology but also for development of effective immunotherapies against unwanted T cell responses in allergy, autoimmunity and cancer.

High lib mRNA expression in breast carcinomas.

Lib, first identified as a novel beta-amyloid responsive gene in rat astrocytes, has an extracellular domain of 15 leucine-rich repeats (LRRs) followed by a transmembrane domain and a short cytoplasmic region. It is a distinctly inducible gene and is thought to play a key role in inflammatory states via the LRR extracellular motif, an ideal structural framework for protein-protein and protein-matrix interactions. To evaluate potential roles of Lib, we screened various tumors for Lib expression. Lib mRNA expression was high and uniquely expressed in breast tumor tissues, compared to paired normal breast tissues. Lib mRNA was localized in the ductal carcinoma cells and Lib protein displayed a homophilic association on the surface of cultured cells. These data suggest that Lib may play a role in the progression of breast carcinomas and may be a diagnostic marker for breast tumors.

Molecular characterization of the dimer formation of Fcα/µ receptor (CD351).

Fcα/µR (CD351) is an Fc receptor for both IgA and IgM and forms an atypical dimer that is resistant to reduction by 2-mercaptoethanol or boiling. We previously demonstrated that the cytoplasmic portion of Fcα/µR is required for dimer formation and for its efficient cell-surface expression. However, the biochemical nature of these phenomena has not been determined. By using a BW5147 mouse cell line expressing deletion mutants of the cytoplasmic region of Fcα/µR, we found that the region spanning amino acids 504-523 was required for efficient cell-surface expression, whereas the region spanning amino acids 481-490 was required for dimmer formation. Immunoblotting analyses of transfectants simultaneously expressing Flag-tagged Fcα/µR and hemagglutinin-tagged Fcα/µR suggested that Fcα/µR does not form homodimers. Instead, our data suggest that Fcα/µR forms heterodimers with an as-yet-unknown molecule with a molecular weight of 60-70 kDa.

Mitsugumin 29 is transcriptionally induced in senile plaque-associated astrocytes.

Astrocytes associated with beta-amyloid (Aß)-deposited senile plaques are a common neuropathological feature of Alzheimer's disease (AD). There is little doubt that the association of Aß with the major component in the central nervous system cells significantly influences disease progression, however, the molecular mechanisms by which Aß contributes to the astrocyte-mediated neuropathological changes have not been well established. In an effort to identify astrocyte-derived molecules that may be closely associated with exacerbation of AD, we identified a novel Aß-induced rat gene, whose mouse counterpart, mitsugumin 29 (MG29), is known to be involved in intracellular Ca²âº homeostasis in skeletal muscle. To evaluate the roles of human MG29 in AD, we investigated its expression in AD brain. In non-AD brains, MG29 mRNA has been detected in neurons, but not in quiescent astrocytes. On the contrary, in AD brains, MG29 is expressed in activated astrocytes associated with senile plaques, but not expressed in neurons around lesions. Human MG29-transfected cells express the protein in the endoplasmic reticulum and the level of expression is involved in Ca²âº-dependent exocytosis mechanisms. Increased MG29 expression in senile plaques-associated activated astrocytes suggests that intracellular Ca²âº turnovers may be changed in these astrocytes. This might be related in sustained Ca²âº-dependent exocytosis of various transmitters including glutamate, leading to the acceleration of neurodegeneration in the lesion observed in AD.

Expression of Th2-skewed pathology mediators in monocyte-derived type 2 of dendritic cells (DC2).

The information conveyed from dendritic cells (DCs) to naïve CD4(+) T cells has crucial influence on their differentiation toward effector T cells. In an effort to identify DC-derived molecules directly contributing to T cell differentiation, we searched for molecules distinctively expressed between two DC subtypes, which were differentiated from peripheral monocytes by cultivation with GM-CSF (for DC1) or IL-3 (for DC2) in the presence of IL-4 and had the ability to induce naïve T cells to differentiate into Th1 or Th2 cells, respectively. As the first step to address this issue, we subtracted DC1 transcripts from those of DC2 and compiled the gene profile dominantly expressed in DC2, whose products are known to reside in other than the nucleus. Intriguingly, many of them were molecules involved in Th2-skewed disease pathologies, such as FN1, ITGAE, GPNMB, PLAUR, FPRL2, LILRB4, SERPINE1, ALOX15, TBXAS1, NCF2, CCL3, IL1RN, SPARC, and STAB1, suggesting that DCs function not only as antigen presenting cells but also as producers of Th2 pathology specific milieus leading to disease deteriorations. We also found that expressions of CYP27A1, PPAP2B, RSAD2, and ABCC3 were up-regulated in DC2, implying their significant function in Th2-deviated states. The identification of differentially expressed genes between DC subtypes provides new insights into their functions and our comparative gene expression profile will be highly useful for the identification of DC-derived key molecules for T cell differentiation.

Lib, transcriptionally induced in senile plaque-associated astrocytes, promotes glial migration through extracellular matrix.

In an effort to identify astrocyte-derived molecules that may be intimately associated with progression of Alzheimer's disease (AD), Lib, a type I transmembrane protein belonging to leucine-rich repeat superfamily, has been identified as a distinctly inducible gene, responsive to beta-amyloid as well as pro-inflammatory cytokines in astrocytes. To evaluate the roles of Lib in AD, we investigated Lib expression in AD brain. In non-AD brain, Lib mRNA has been detected in neurons but not in quiescent astrocytes. On the contrary, in AD brain, Lib mRNA is expressed in activated astrocytes associated with senile plaques, but not expressed in neurons around lesions. Lib-expressing glioma cells displayed promotion of migration ability through reconstituted extracellular matrix and recombinant Lib protein bound to constituents of extracellular matrix. These observations suggest that Lib may contribute to regulation of cell-matrix adhesion interactions with respect to astrocyte recruitment around senile plaques in AD brain.

A novel member of the leucine-rich repeat superfamily induced in rat astrocytes by beta-amyloid.

beta-Amyloid (Abeta) deposition and senile plaque-associated astrocytes are common neuropathological features of Alzheimer's disease (AD). Although the molecular mechanisms by which Abeta contributes to the progression of neuropathologic changes have not been established entirely, there is little doubt that the association of Abeta with astrocytes, the predominant cell type in brain, has significant influence on exacerbation of the disease. In an effort to identify key molecules involved in AD, we investigated Abeta-responsive genes using rat astrocytes. In this study, we identified a novel Abeta-induced rat gene, designated as Lib, encoding a type I transmembrane protein with an extracellular domain that contains fifteen leucine-rich repeats (LRRs). Human counterpart of rat Lib is located on chromosome 3q29 and human Lib mRNA found in particularly placenta. Lib mRNA levels in rat C6 astrocytoma cells can be increased by pro-inflammatory cytokines and the rat Lib-transfected cells express Lib protein on the cell surfaces. Lib appears to be a member of the LRR superfamily which is involved in cell-cell and/or -extracellular matrix interactions including adhesion or target recognition in neuroinflammatory states.