Glucocorticoid withdrawal and glucocorticoid-induced adrenal insufficiency: Study protocol of the randomized controlled «TOASST" (Taper Or Abrupt Steroid STop) multicenter trial.

BACKGROUND: Despite the widespread use of glucocorticoids in inflammatory and autoimmune disorders, there is uncertainty about the safe cessation of long-term systemic treatment, as data from prospective trials are largely missing. Due to potential disease relapse or glucocorticoid-induced hypocortisolism, the drug is often tapered to sub-physiological doses rather than stopped when the underlying disease is clinically stable, increasing the cumulative drug exposure. Conversely, the duration of exposure to glucocorticoids should be minimized to lower the risk of side effects. METHODS: We designed a multicenter, randomized, triple-blinded, placebo-controlled trial to test the clinical noninferiority of abrupt glucocorticoid stop compared to tapering after ≥28 treatment days with ≥420 mg cumulative and ≥7.5 mg mean daily prednisone-equivalent dose. 573 adult patients treated systemically for various disorders will be included after their underlying disease has been stabilized. Prednisone in tapering doses or matching placebo is administered over 4 weeks. A 250 mg ACTH-test, the result of which will be revealed a posteriori, is performed at study inclusion; all patients are instructed on glucocorticoid stress cover dosing. Follow-up is for 6 months. The composite primary outcome measure is time to hospitalization, death, initiation of unplanned systemic glucocorticoid therapy, or adrenal crisis. Secondary outcomes include the individual components of the primary outcome, cumulative glucocorticoid doses, signs and symptoms of hypocortisolism, and the performance of the ACTH test in predicting the clinical outcome. Cox proportional hazard, linear, and logistic regression models will be used for statistical analysis. CONCLUSION: This trial aims to demonstrate the clinical noninferiority and safety of abrupt treatment cessation after ≥28 days of systemic glucocorticoid therapy in patients with stabilized underlying disease. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT03153527; EUDRA-CT: 2020-005601-48 https://clinicaltrials.gov/ct2/show/NCT03153527?term=NCT03153527&draw=2&rank=1.

Production cell analysis and compound-based boosting of small extracellular vesicle secretion using a generic and scalable production platform.

Extracellular vesicles (EVs) are a novel format of advanced therapeutical medicinal products (ATMPs). They can act regenerative or immune-modulatory as cell therapy substitutes or as a platform for designer exosomes. The biotechnological production of therapeutic EVs is still very much uncharted territory so standardized host cells, production setups, and isolation methods are not yet implemented. In this work, we present a tangential flow filtration (TFF) and fast-performance liquid chromatography (FPLC)-based size exclusion chromatography (SEC) purification setup that is compatible for industry applications. Moreover, we evaluated a series of potential host cell lines regarding their EV productivity, characteristics, and biological functionality. It was found that telomerase-immortalized Wharton's jelly mesenchymal stromal cells (WJ-MSC/TERT273) secrete high amounts of EVs per cell with regenerative capabilities. On the other hand, Cevec's amniocyte producer cells® (CAP®) and human embryonic kidney (HEK293) suspension cells are suitable platforms for designer EVs with high yields. Finally, we aimed to boost the EV secretion of HEK293 cells via chemical adjuvants and verified four compounds that heighten cellular EV secretion in a presumably cAMP-dependent manner. A combination of fenoterol, iodoacetamide, and dinitrophenol increased the EV yield in HEK293 cells threefold and cellular secretion rate fivefold.

Human CAP cells represent a novel source for functional, miRNA-loaded exosome production.

Exosomes represent a promising delivery tool for nucleic acid-based pharmaceuticals. They are highly suitable for transporting therapeutic miRNAs to tumor cells, due to their natural membrane components. Further, exosomes are capable of effectively protecting nucleic acids against ribonucleases and enable the delivery of their content through cell membranes. However, no suitable production host for miRNA containing exosomes of non-tumorigenic origin has yet been identified. In this study we engineered an immortalised human amniocyte cell line (CAP® cells), whose exosomes were enriched and characterised. The cell line modifications not only enabled the production of GFP-labelled but also pro-apoptotic miRNA containing exosomes without negative influence on host cell growth. Furthermore, we demonstrated that pro-apoptotic miRNA containing CAP exosomes are taken up by ovarian cancer cells. Strikingly, delivery of functional exosomal miRNA led to downregulation of several reported target genes in the treated tumor cells. In summary, we revealed CAP cells of non-tumorigenic origin as a novel and efficient exosome production host with the potential to produce functional miRNA-loaded exosomes.

Induction of apoptosis in ovarian cancer cells by miR-493-3p directly targeting AKT2, STK38L, HMGA2, ETS1 and E2F5.

Apoptosis is a form of directed programmed cell death with a tightly regulated signalling cascade for the destruction of single cells. MicroRNAs (miRNAs) play an important role as fine tuners in the regulation of apoptotic processes. MiR-493-3p mimic transfection leads to the induction of apoptosis causing the breakdown of mitochondrial membrane potential and the activation of Caspases resulting in the fragmentation of DNA in several ovarian carcinoma cell lines. Ovarian cancer shows with its pronounced heterogeneity a very high death-to-incidence ratio. A target gene analysis for miR-493-3p was performed for the investigation of underlying molecular mechanisms involved in apoptosis signalling pathways. Elevated miR-493-3p levels downregulated the mRNA and protein expression levels of Serine/Threonine Kinase 38 Like (STK38L), High Mobility Group AT-Hook 2 (HMGA2) and AKT Serine/Threonine Kinase 2 (AKT2) by direct binding as demonstrated by luciferase reporter assays. Notably, the protein expression of RAF1 Proto-Oncogene, Serine/Threonine Kinase (RAF1) was almost completely downregulated by miR-493-3p. This interaction, however, was indirect and regulated by STK38L phosphorylation. In addition, RAF1 transcription was diminished as a result of reduced transcription of ETS proto-oncogene 1 (ETS1), another direct target of miR-493-3p. Taken together, our observations have uncovered the apoptosis inducing potential of miR-493-3p through its regulation of multiple target genes participating in the extrinsic and intrinsic apoptosis pathway.

MiR-744-5p inducing cell death by directly targeting HNRNPC and NFIX in ovarian cancer cells.

MicroRNAs (miRNAs) play an important role in the regulation of gene expression. The binding to target messenger RNAs (mRNAs) results in mRNA cleavage or inhibition of the translational machinery leading to decreased protein levels. Various signalling pathways, including apoptosis are modulated by miRNAs. Here, we investigated the role of miR-744-5p in apoptosis signalling in ovarian cancer cell lines. MiR-744-5p expression was reduced in the cancer cell lines independent of the host gene MAP2K4. Overexpression of miR-744-5p activated the intrinsic apoptotic pathway in SKOV3, OVCAR3 and Cisplatin resistant (A2780-cis) and non-resistant A2780 cells leading to cell death. Notably, miR-744-5p overexpression together with Carboplatin treatment led to at least additive pro-apoptotic effects. Investigation of the apoptotic signalling pathways mediated by miR-744-5p revealed that its elevated expression directly downregulated mRNA and protein expression of nuclear factor I X (NFIX) and heterogeneous nuclear ribonucleoprotein C (HNRNPC). HNRNPC caused diminished miR-21 expression and AKT phosphorylation, while NFIX decreased Bcl2 levels, leading to the detected pro-apoptotic effects. Finally, Kaplan-Meier-Plots showed a prolonged median disease-free survival in ovarian serous cystadenocarcinoma patients with high miR-744 expression.

miR-217-5p induces apoptosis by directly targeting PRKCI, BAG3, ITGAV and MAPK1 in colorectal cancer cells.

Apoptosis is a genetically directed process of programmed cell death. A variety of microRNAs (miRNAs), endogenous single-stranded non-coding RNAs of about 22 nucleotides in length have been shown to be involved in the regulation of the intrinsic or extrinsic apoptotic pathways. There is increasing evidence that the aberrant expression of miRNAs plays a causal role in the development of diseases such as cancer. This makes miRNAs promising candidate molecules as therapeutic targets or agents. MicroRNA (miR)-217-5p has been implicated in carcinogenesis of various cancer entities, including colorectal cancer. Here, we analyzed the pro-apoptotic potential of miR-217-5p in a variety of colorecatal cancer cell lines showing that miR-217-5p mimic transfection led to the induction of apoptosis causing the breakdown of mitochondrial membrane potential, externalization of phosphatidylserine, activation of caspases and fragmentation of DNA. Furthermore, elevated miR-217-5p levels downregulated mRNA and protein expression of atypical protein kinase c iota type I (PRKCI), BAG family molecular chaperone regulator 3 (BAG3), integrin subunit alpha v (ITGAV) and mitogen-activated protein kinase 1 (MAPK1). A direct miR-217-5p mediated regulation to those targets was shown by repressed luciferase activity of reporter constructs containing the miR-217-5p binding sites in the 3' untranslated region. Taken together, our observations have uncovered the apoptosis-inducing potential of miR-217-5p through its regulation of multiple target genes involved in the ERK-MAPK signaling pathway by regulation of PRKCI, BAG3, ITGAV and MAPK1.

The Immune Adaptor SLP-76 Binds to SUMO-RANGAP1 at Nuclear Pore Complex Filaments to Regulate Nuclear Import of Transcription Factors in T Cells.

While immune cell adaptors regulate proximal T cell signaling, direct regulation of the nuclear pore complex (NPC) has not been reported. NPC has cytoplasmic filaments composed of RanGAP1 and RanBP2 with the potential to interact with cytoplasmic mediators. Here, we show that the immune cell adaptor SLP-76 binds directly to SUMO-RanGAP1 of cytoplasmic fibrils of the NPC, and that this interaction is needed for optimal NFATc1 and NF-κB p65 nuclear entry in T cells. Transmission electron microscopy showed anti-SLP-76 cytoplasmic labeling of the majority of NPCs in anti-CD3 activated T cells. Further, SUMO-RanGAP1 bound to the N-terminal lysine 56 of SLP-76 where the interaction was needed for optimal RanGAP1-NPC localization and GAP exchange activity. While the SLP-76-RanGAP1 (K56E) mutant had no effect on proximal signaling, it impaired NF-ATc1 and p65/RelA nuclear entry and in vivo responses to OVA peptide. Overall, we have identified SLP-76 as a direct regulator of nuclear pore function in T cells.

Diverse mechanisms regulate the surface expression of immunotherapeutic target ctla-4.

T-cell co-receptor cytotoxic T-cell antigen-4 (CTLA-4) is a critical inhibitory regulator of T-cell immunity and antibody blockade of the co-receptor has been shown to be effective in tumor immunotherapy. Paradoxically, the majority of CTLA-4 is located in intracellular compartments from where it is transported to the cell surface and rapidly internalized. The intracellular trafficking pathways that control transport of the co-receptor to the cell surface ensures the appropriate balance of negative and positive signaling for a productive immune response with minimal autoimmune disorders. It will also influence the degree of inhibition and the potency of antibody checkpoint blockade in cancer immunotherapy. Current evidence indicates that the mechanisms of CTLA-4 transport to the cell surface and its residency are multifactorial involving a combination of immune cell-specific adapters such as TRIM and LAX, the small GTPase Rab8 as well as generic components such as ARF-1, phospholipase D, and the heterotetrameric AP1/2 complex. This review covers the recent developments in our understanding of the processes that control the expression of this important co-inhibitory receptor for the modulation of T-cell immunity. Interference with the processes that regulate CTLA-4 surface expression could provide an alternate therapeutic approach in the treatment of cancer and autoimmunity.

CTLA-4 activation of phosphatidylinositol 3-kinase (PI 3-K) and protein kinase B (PKB/AKT) sustains T-cell anergy without cell death.

The balance of T-cell proliferation, anergy and apoptosis is central to immune function. In this regard, co-receptor CTLA-4 is needed for the induction of anergy and tolerance. One central question concerns the mechanism by which CTLA-4 can induce T-cell non-responsiveness without a concurrent induction of antigen induced cell death (AICD). In this study, we show that CTLA-4 activation of the phosphatidylinositol 3-kinase (PI 3-K) and protein kinase B (PKB/AKT) sustains T-cell anergy without cell death. CTLA-4 ligation induced PI 3K activation as evidenced by the phosphorylation of PKB/AKT that in turn inactivated GSK-3. The level of activation was similar to that observed with CD28. CTLA-4 induced PI 3K and AKT activation also led to phosphorylation of the pro-apoptotic factor BAD as well as the up-regulation of BcL-XL. In keeping with this, CD3/CTLA-4 co-ligation prevented apoptosis under the same conditions where T-cell non-responsiveness was induced. This effect was PI 3K and PKB/AKT dependent since inhibition of these enzymes under conditions of anti-CD3/CTLA-4 co-ligation resulted in cell death. Our findings therefore define a mechanism by which CTLA-4 can induce anergy (and possibly peripheral tolerance) by preventing the induction of cell death.

CTLA-4 trafficking and surface expression.

The T-cell co-receptor cytotoxic T-cell antigen 4 (CTLA-4) has a strong inhibitory role as shown by the lymphoproliferative phenotype of CTLA-4-deficient mice. Despite its potent effects on T-cell function, CTLA-4 is primarily an intracellular antigen whose surface expression is tightly regulated by restricted trafficking to the cell surface and rapid internalisation. Recently, several signalling molecules such as Trim, PLD, ARF-1 and TIRC7 have been described to be involved in the transport of CTLA-4 to the cell surface. Minor changes in surface expression levels have major effects on the outcome of T-cell activation. Optimal regulation of CTLA-4 surface expression is crucial for the balance of stimulatory and inhibitory signals to maximize protective immune responses while maintaining immunological tolerance and preventing autoimmunity.

Adaptor SKAP-55 binds p21 activating exchange factor RasGRP1 and negatively regulates the p21-ERK pathway in T-cells.

While the adaptor SKAP-55 mediates LFA-1 adhesion on T-cells, it is not known whether the adaptor regulates other aspects of signaling. SKAP-55 could potentially act as a node to coordinate the modulation of adhesion with downstream signaling. In this regard, the GTPase p21(ras) and the extracellular signal-regulated kinase (ERK) pathway play central roles in T-cell function. In this study, we report that SKAP-55 has opposing effects on adhesion and the activation of the p21(ras) -ERK pathway in T-cells. SKAP-55 deficient primary T-cells showed a defect in LFA-1 adhesion concurrent with the hyper-activation of the ERK pathway relative to wild-type cells. RNAi knock down (KD) of SKAP-55 in T-cell lines also showed an increase in p21(ras) activation, while over-expression of SKAP-55 inhibited activation of ERK and its transcriptional target ELK. Three observations implicated the p21(ras) activating exchange factor RasGRP1 in the process. Firstly, SKAP-55 bound to RasGRP1 via its C-terminus, while secondly, the loss of binding abrogated SKAP-55 inhibition of ERK and ELK activation. Thirdly, SKAP-55-/- primary T-cells showed an increased presence of RasGRP1 in the trans-Golgi network (TGN) following TCR activation, the site where p21(ras) becomes activated. Our findings indicate that SKAP-55 has a dual role in regulating p21(ras)-ERK pathway via RasGRP1, as a possible mechanism to restrict activation during T-cell adhesion.

CD28 and Grb-2, relative to Gads or Grap, preferentially co-operate with Vav1 in the activation of NFAT/AP-1 transcription.

The co-receptor CD28 binds to several intracellular proteins including PI3 kinase, Grb-2, Gads and ITK. Grb-2 and PI3 kinase binding has been mapped to the pYMNM motif within the cytoplasmic tail of CD28 and has been shown to play a role in co-stimulation. In this study, we demonstrate that amongst the Grb-2 family adapter proteins, CD28 precipitated Grb-2 and specifically co-operated in the up-regulation of NFAT/AP-1 transcription. By contrast, Gads and Grap either failed or only weakly collaborated with CD28 ligation. Further, the loss of Grb-2 binding interferes with the ability of Vav1 to co-operate with CD28. Anti-CD28 ligation alone was capable for co-operating with Grb-2 or Grb-2-Vav1. Our findings define a pathway involving CD28 binding to Grb-2 and its co-operativity with Vav1 in the regulation of T-cell co-stimulation.

TCR/CD3 mediated stop-signal is decoupled in T-cells from Ctla4 deficient mice.

CTLA-4 is a co-receptor that plays a pivotal role in regulating the threshold for T-cell activation. We recently reported that CTLA-4 ligation can over-ride the stop-signal induced by anti-CD3 ligation [Schneider H, Downey J, Smith A, Zinselmeyer BH, Rush C, Brewer JM, et al. Reversal of the TCR stop-signal by CTLA-4. Science 2006;313:1972]. While these studies compared CTLA-4 positive and negative T-cells from normal mice, little is known regarding the behaviour of T-cells from diseased Ctla4 deficient mice with auto-proliferative disease. In this study, we show that while activated wild-type and Ctla-4-/- T-cells have similar rates of motility, Ctla-4-/- T-cells show a marked resistance to the induction of a stop-signal by anti-CD3 ligation. By contrast, T-cells from normal mice and CD28 deficient mice underwent a normal slowing of motility in response to anti-CD3 ligation. Our findings identify a fundamental difference between normal versus CTLA-4-/- T-cells from diseased mice in the regulation of motility by anti-CD3 ligation. This dysregulation of motility may contribute to the tissue infiltration and the autoimmune disorder observed in Ctla-4-/- mice.

CTLA-4 disrupts ZAP70 microcluster formation with reduced T cell/APC dwell times and calcium mobilization.

CTLA-4 is a co-receptor that modulates the threshold of T cell activation and autoimmunity. We previously showed that CTLA-4 reverses the TCR-mediated stop signal needed for T cell/APC interactions [Schneider et al., Science 2006, 313: 1972]. In this study, using a different T cell system, we show that CTLA-4 expression changed the behavior of T8.1 T cells by reducing the contact time between T cell and APC, preventing re-inforced contacts, and reducing the contact area at the immunological synapse. This led to a major reduction in Ca(2+) influx/mobilization and interleukin-2 production. Further, anti-CD3/CTLA-4 increased T cell motility on antibody-coated glass slides, concurrent with an abrogation of ZAP70 microcluster formation. Our findings further support a role for CTLA-4 in limiting the interaction between T cell and APC that is needed for optimal activation.

T cell receptor-interacting molecule acts as a chaperone to modulate surface expression of the CTLA-4 coreceptor.

The costimulatory molecule CTLA-4 is a potent downregulator of T cell responses. Although localized mostly in intracellular compartments, little is understood regarding the mechanism that regulates its transport to the cell surface. In this study, we demonstrated that the adaptor TRIM (T cell receptor-interacting molecule) bound to CTLA-4 in the trans Golgi network (TGN) and promoted transport of CTLA-4 to the surface of T cells. Increased TRIM expression augmented surface CTLA-4 expression, and pulse-chase analysis showed a more rapid transport of CTLA-4 to the cell surface. A reduction of TRIM expression by small hairpin RNAs reduced the expression of surface CTLA-4. This resulted in a more localized pattern of CTLA-4 in the TGN. Altered CTLA-4 expression by TRIM was accompanied by corresponding changes in coreceptor-mediated effects on cytokine production and proliferation. Our findings identify a role for TRIM as a chaperone in regulating CTLA-4 expression and function by enhancing CTLA-4 transport to the surface of T cells.

Reversal of the TCR stop signal by CTLA-4.

The coreceptor cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) is pivotal in regulating the threshold of signals during T cell activation, although the underlying mechanism is still not fully understood. Using in vitro migration assays and in vivo two-photon laser scanning microscopy, we showed that CTLA-4 increases T cell motility and overrides the T cell receptor (TCR)-induced stop signal required for stable conjugate formation between T cells and antigen-presenting cells. This event led to reduced contact periods between T cells and antigen-presenting cells that in turn decreased cytokine production and proliferation. These results suggest a fundamentally different model of reverse stop signaling, by which CTLA-4 modulates the threshold for T cell activation and protects against autoimmunity.

TcR and TcR-CD28 engagement of protein kinase B (PKB/AKT) and glycogen synthase kinase-3 (GSK-3) operates independently of guanine nucleotide exchange factor VAV-1.

TcRzeta/CD3 and TcRzeta/CD3-CD28 signaling requires the guanine nucleotide exchange factor (GEF) Vav-1 as well as the activation of phosphatidylinositol 3-kinase, protein kinase B (PKB/AKT), and its inactivation of glycogen synthase kinase-3 (GSK-3). Whether these two pathways are connected or operate independently of each other in T-cells has been unclear. Here, we report that anti-CD3 and anti-CD3/CD28 can induce PKB and GSK-3alpha phosphorylation in the Vav-1(-/-) Jurkat cell line J. Vav.1 and in primary CD4-positive Vav-1(-/-) T-cells. Reduced GSK-3alpha phosphorylation was observed in Vav-1,2,3(-/-) T-cells together with a complete loss of FOXO1 phosphorylation. Furthermore, PKB and GSK-3 phosphorylation was unperturbed in the presence of GEF-inactive Vav-1 that inhibited interleukin-2 gene activation and a form of Src homology 2 domain-containing lymphocytic protein of 76-kDa (SLP-76) that is defective in binding to Vav-1. The pathway also was intact under conditions of c-Jun N-terminal kinase (JNK) inhibition and disruption of the actin cytoskeleton by cytochalasin D. Both events are down-stream targets of Vav-1. Overall, our findings indicate that the TcR and TcR-CD28 driven PKB-GSK-3 pathway can operate independently of Vav-1 in T-cells.

CTLA-4 up-regulation of lymphocyte function-associated antigen 1 adhesion and clustering as an alternate basis for coreceptor function.

Although cytotoxic T lymphocyte antigen-4 (CTLA-4) negatively regulates T cell activation, the full range of functions mediated by this coreceptor has yet to be established. In this study, we report the surprising finding that CTLA-4 engagement by soluble antibody or CD80 potently up-regulates lymphocyte function-associated antigen 1 (LFA-1) adhesion to intercellular adhesion molecule-1 (ICAM-1) and receptor clustering concurrent with IL-2 inhibition. This effect was also observed with CTLA-4 ligation and not with other coreceptors. T cell antigen receptor (TcR)-induced lymphocyte function-associated antigen 1 function was also dependent on CTLA-4 expression as observed with reduced adhesion/clustering on CTLA-4(-/-) primary T cells. CTLA-4 up-regulated adhesion was mediated by regulator for cell adhesion and polarization type 1 (Rap-1) as shown by anti-CTLA-4-induced Rap-1 activation as well as Rap-1-N17 blockade and Rap-1-V12 mimicry of adhesion/clustering. Our findings identify a potent role for CTLA-4 in directing integrin adhesion and provide an alternate mechanism to account for aspects of CTLA-4 function in T cell immunity.

Unifying concepts in CD28, ICOS and CTLA4 co-receptor signalling.

Many studies have shown the central importance of the co-receptors CD28, inducible costimulatory molecule (ICOS) and cytotoxic T lymphocyte antigen 4 (CTLA4) in the regulation of many aspects of T-cell function. CD28 and ICOS have both overlapping and distinct functions in the positive regulation of T-cell responses, whereas CTLA4 negatively regulates the response. The signalling pathways that underlie the function of each of the co-receptors indicate their shared and unique properties and provide compelling hints of functions that are as yet uncovered. Here, we outline the shared and distinct signalling events that are associated with each of the co-receptors and provide unifying concepts that are related to signalling functions of these co-receptors.