Maria Felicia Soluri

Engineering mammalian cell factories with SINEUP noncoding RNAs to improve translation of secreted proteins

Whenever the function of a recombinant protein depends on post-translational processing, mammalian cells become an indispensable tool for their production. This is particularly true for biologics and therapeutic monoclonal antibodies (MAbs). Despite some drawbacks, Chinese Hamster Ovary (CHO) cells are the workhorse for MAbs production in academia and industry. Several methodologies have been adopted to improve expression and stability, including methods based on selective pressure or cell engineering. We have previously identified SINEUPs as a new functional class of natural and synthetic long non-coding RNAs that through the activity of an inverted SINEB2 element are able to promote translation of partially overlapping sense coding mRNAs. Here we show that by taking advantage of their modular structure, synthetic SINEUPs can be designed to increase production of secreted proteins. Furthermore, by experimentally validating antisense to elastin (AS-eln) RNA as a natural SINEUP, we show that SINEUP-mediated control may target extracellular proteins. These results lead us to propose synthetic SINEUPs as new versatile tools to optimize production of secreted proteins in manufacturing pipelines and natural SINEUPs as new regulatory RNAs in the secretory pathways.

Thrombin Cleavage of Osteopontin Modulates Its Activities in Human Cells In Vitro and Mouse Experimental Autoimmune Encephalomyelitis In Vivo

Osteopontin is a proinflammatory cytokine and plays a pathogenetic role in multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE), by recruiting autoreactive T cells into the central nervous system. Osteopontin functions are modulated by thrombin cleavage generating N- and C-terminal fragment, whose individual roles are only partly known. Published data are difficult to compare since they have been obtained with heterogeneous approaches. Interestingly, thrombin cleavage of osteopontin unmasks a cryptic domain of interaction with α 4 β 1 integrin that is the main adhesion molecule involved in lymphocyte transmigration to the brain and is the target for natalizumab, the most potent drug preventing relapses. We produced recombinant osteopontin and its N- and C-terminal fragments in an eukaryotic system in order to allow their posttranslational modifications. We investigated, in vitro, their effect on human cells and in vivo in EAE. We found that the osteopontin cleavage plays a key role in the function of this cytokine and that the two fragments exert distinct effects both in vitro and in vivo. These findings suggest that drugs targeting each fragment may be used to fine-tune the pathological effects of osteopontin in several diseases.

Variations of the UNC13D Gene in Patients with Autoimmune Lymphoproliferative Syndrome

Autoimmune lymphoproliferative syndrome (ALPS) is caused by genetic defects decreasing Fas function and is characterized by lymphadenopathy/splenomegaly and expansion of CD4/CD8 double-negative T cells. This latter expansion is absent in the ALPS variant named Dianzani Autoimmune/lymphoproliferative Disease (DALD). In addition to the causative mutations, the genetic background influences ALPS and DALD development. We previously suggested a disease-modifying role for the perforin gene involved in familial hemophagocytic lymphohistiocytosis (FHL). The UNC13D gene codes for Munc13-4, which is involved in perforin secretion and FHL development, and thus, another candidate for a disease-modifying role in ALPS and DALD. In this work, we sequenced UNC13D in 21 ALPS and 20 DALD patients and compared these results with sequences obtained from 61 healthy subjects and 38 multiple sclerosis (MS) patients. We detected four rare missense variations in three heterozygous ALPS patients carrying p.Cys112Ser, p.Val781Ile, and a haplotype comprising both p.Ile848Leu and p.Ala995Pro. Transfection of the mutant cDNAs into HMC-1 cells showed that they decreased granule exocytosis, compared to the wild-type construct. An additional rare missense variation, p.Pro271Ser, was detected in a healthy subject, but this variation did not decrease Munc13-4 function. These data suggest that rare loss-of-function variations of UND13D are risk factors for ALPS development.

Osteopontin Bridging Innate and Adaptive Immunity in Autoimmune Diseases

Osteopontin (OPN) regulates the immune response at multiple levels. Physiologically, it regulates the host response to infections by driving T helper (Th) polarization and acting on both innate and adaptive immunity; pathologically, it contributes to the development of immune-mediated and inflammatory diseases. In some cases, the mechanisms of these effects have been described, but many aspects of the OPN function remain elusive. This is in part ascribable to the fact that OPN is a complex molecule with several posttranslational modifications and it may act as either an immobilized protein of the extracellular matrix or a soluble cytokine or an intracytoplasmic molecule by binding to a wide variety of molecules including crystals of calcium phosphate, several cell surface receptors, and intracytoplasmic molecules. This review describes the OPN structure, isoforms, and functions and its role in regulating the crosstalk between innate and adaptive immunity in autoimmune diseases.

Differential induction of IL-17, IL-10, and IL-9 in human T helper cells by B7h and B7.1

ICOS and CD28 are expressed by T cells and are involved in costimulation of cytokine production in T helper (TH) cells. ICOS binds B7h expressed by several cell types, whereas CD28 binds B7.1 and B7.2 expressed by activated antigen presenting cells. This work investigated the role of B7h and B7.1 in TH17 and TH9 cell differentiation by assessing activity of recombinant B7h-Fc and B7.1-Fc on human naïve TH cells activated in the presence of different combinations of exogenous cytokines. In the presence of TGF-β1 and IL-1β (TH17 promoting condition), B7h-Fc was more effective than B7.1-Fc in inducing IL-17A and IL-10 secretion, whereas B7.1-Fc was more effective in inducing IL-17F. Dual costimulation with B7h-Fc and B7.1-Fc displayed an intermediate pattern with predominance of IL-17F over IL-17A, secretion of high levels of IL-10, and secretion of IL-9 levels lower than those induced by B7.1-Fc alone. In the presence of TGF-β1 and IL-4 (TH9 promoting condition), B7h-Fc induced IL-17A only, whereas B7.1-Fc induced also IL-17F, IL-10, and high levels of IL-9. Experiments on memory TH cells showed that B7h-Fc mainly supported secretion of IL-17A and IL-10, whereas B7.1-Fc supported secretion of IL-17A, IL-17F, IL-10, and IL-9. These data indicate that B7h and B7.1 play different roles in modulation of TH17 and TH9 differentiation. This plasticity might be important in the immune response to pathogens and tumors, and in the development of autoimmune diseases, and should be taken in consideration in designing of immunotherapeutic protocols triggering ICOS or CD28.

Triggering of B7h by the ICOS modulates maturation and migration of monocyte-derived dendritic cells

B7h, expressed by several cell types, binds ICOS expressed by activated T cells. We have previously shown that B7h triggering by ICOS-Fc inhibits human endothelial cell adhesiveness. This work investigated the effect of ICOS-Fc on human monocyte-derived dendritic cells (DCs). We found that DCs matured with LPS in the presence of ICOS-Fc (mDCsICOS) produced greater amounts of IL-23 and IL-10, and promoted a higher secretion of IL-17A and IL-17F in MLCs than did those DCs matured with LPS alone (mDCs). Moreover, mDCsICOS pulsed with the keyhole limpet hemocyanin Ag during the maturation phase were better stimulators of Ag-specific MHC class I–, but not class II–restricted T cells than mDCs. This was probably due to promotion of cross-presentation because it was not detected when the Flu-MA58–66 Ag was directly loaded on already matured DCs and mDCsICOS. Finally, ICOS-Fc inhibited the adhesion of both immature DCs and mDCs to vascular and lymphoid endothelial cells, their migratory activity, and the expression of the Rac-1 activator β-Pix involved in cell motility. These data suggest that B7h stimulation modulates DC function with effects on their maturation and recruitment into tissues. This opens a novel view on the use of interactors of the ICOS:B7h system as immunomodulatory drugs.

The -346T polymorphism of the SH2D1A gene is a risk factor for development of autoimmunity/lymphoproliferation in males with defective Fas function.

Inherited defects decreasing function of the Fas death receptor cause autoimmune lymphoproliferative syndrome (ALPS) and its variant Dianzani autoimmune lymphoproliferative disease (DALD). Since a deleterious mutation of the SH2D1A gene protects MRLlpr/lpr mice from ALPS development, we investigated the role of SH2D1A, located in the X chromosome, in 51 patients with ALPS or DALD by mutational screening of coding and regulative sequences. Allelic frequency of the -346C>T polymorphism was different in male patients and controls (-346T: 61% vs 36%, p = 0.01), with similar frequencies in ALPS and DALD. By contrast, no differences were found among females or between the controls and patients with multiple sclerosis (229 males, 157 females). Further analyses showed that -346C was a methylation site in CD8(+) T and natural killer cells, and SH2D1A expression was higher in -346T than in -346C males. Finally, in vitro-activated T cells from -346T males produced lower amounts of interferon-γ than those from -346C males. These data suggest that -346T is a predisposing factor for ALPS and DALD in males possibly because of its effect on SAP expression influencing the T-cell response.

Tissue transglutaminase (TG2) enables survival of human malignant pleural mesothelioma cells in hypoxia

Malignant pleural mesothelioma (MPM) is an aggressive tumor linked to environmental/occupational exposure to asbestos, characterized by the presence of significant areas of hypoxia. In this study, we firstly explored the expression and the role of transglutaminase 2 (TG2) in MPM cell adaptation to hypoxia. We demonstrated that cells derived from biphasic MPM express the full-length TG2 variant at higher levels than cells derived from epithelioid MPM and normal mesothelium. We observed a significant induction of TG2 expression and activity when cells from biphasic MPM were grown as a monolayer in chronic hypoxia or packed in spheroids, where the presence of a hypoxic core was demonstrated. We described that the hypoxic induction of TG2 was HIF-2 dependent. Importantly, TGM2-v1 silencing caused a marked and significant reduction of MPM cell viability in hypoxic conditions when compared with normoxia. Notably, a TG2-selective irreversible inhibitor that reacts with the intracellular active form of TG2, but not a non-cell-permeable inhibitor, significantly compromised cell viability in MPM spheroids. Understanding the expression and function of TG2 in the adaptation to the hypoxic environment may provide useful information for novel promising therapeutic options for MPM treatment.