Introduction Granulocyte-colony stimulating factor (G-CSF) administration is known to induce severe vaso-occlusive crisis (VOC) in patients with sickle cell disease (SCD). G-CSF and neutrophils rise acutely during VOC, whereas an elevated circulating neutrophil count at steady state is a prognostic factor of SCD severity. G-CSF is induced by many inflammatory triggers, including lipopolysaccharides (LPS), tumor necrosis factor alpha (TNFα) and toll-like receptor 4 activation, which provoke VOC in SCD mice. Importantly, G-CSF induces neutrophil extracellular traps (NETs) formation and activates platelets and monocytes, leading to endothelial cell activation and thrombo-inflammation in SCD. We report the first preclinical evidence validating G-CSF receptor (G-CSFR) blockade as a therapeutic target to prevent VOC in SCD. Methods Townes knock-in humanized SCD mice were exposed to hypoxia-reoxygenation (HR) or intravenously (IV) challenged with oxy-hemoglobin (oxy-Hb), LPS, or G-CSF to induce VOC. Some mice were pretreated IV with VR81, an anti-mouse G-CSFR monoclonal antibody (mAb), prior to VOC induction. Blood flow and NETs formation were assessed by intravital microscopy of the skin and lungs. Liver and lung tissues were examined for inflammation by Western blots and immunofluorescence staining, respectively. Gene expression analyses were performed on transcriptomic datasets from the blood of SCD patients with/without VOC and healthy humans with/without treatment with G-CSF and/or CSL324, an anti-human G-CSFR mAb under development. Results A single IV dose of VR81 in mice had a half-life of ~7 days. Full receptor occupancy in neutrophils was immediate, maintained for at least 1 week, and confirmed by the complete inhibition of phosphorylated STAT3, the signal transduction mediator of G-CSFR signaling. G-CSF administered IV to SCD mice dose-dependently induced microvascular vaso-occlusion in the skin. Vaso-occlusion in the skin was prevented by VR81 administration IV 1 hour or 7 days prior to VOC induction with G-CSF, HR, or oxy-Hb. Importantly, VR81 resolved skin vaso-occlusion in SCD mice when administered 40 minutes after VOC induction. Biomarker analysis revealed several effects caused by G-CSFR blockade. In the liver, expression of pro-inflammatory endothelial adhesion molecules VCAM-1, ICAM-1, and E-selectin and nuclear factor-ĸB-phospho-p65 were markedly decreased, while expression of anti-inflammatory heme oxygenase-1 and nuclear localization of nuclear factor erythroid 2-related factor 2 were increased. Intravital microscopy in the lungs revealed that VR81 pretreatment also prevented neutrophil-platelet aggregate-dependent lung vaso-occlusion in oxy-Hb challenged SCD mice, with a reduced number of NETs within the lung and blood circulation. In the lungs, the number of neutrophils, the expression of pro-adhesive Weibel-Palade body P-selectin, and von Willebrand factor were decreased. Long-term administration of VR81 once per week for 5 months reduced signs of chronic organ injury. The weight of the right ventricle was normalized, suggesting a possible reduction in pulmonary hypertension. The white blood cell count was also reduced. Transcriptomic analyses support the translatability of G-CSFR blockade to SCD patients. We previously published a gene signature of differentially expressed genes in blood after G-CSF administration to healthy humans. In SCD patients, this G-CSF gene signature was significantly enriched during VOC vs. baseline (p-value < 10-3) when analyzed using microarray and RNA-Seq datasets. Conclusions Literature and our data strongly support the critical role of G-CSF in thrombo-inflammatory pathobiology in VOC. Because G-CSF activates multiple inflammatory pathways that trigger vaso-occlusion, G-CSFR is a suitable target for drug intervention to prevent VOC. Our data suggest multiple anti-inflammatory effects in SCD, including prevention of NET formation, which provide a plausible mechanism of action for G-CSFR blockade. The known biology of NETs aligns with a role for G-CSFR in SCD. The NETs formation and their rise during VOC have been confirmed from the literature for patients with SCD. CSL324 was safe and well tolerated in repeat-dose toxicological studies in non-human primates and has completed a phase 1 trial, with satisfactory safety (some risk of transient neutropenia) and pharmacokinetic profiles. A phase 2 trial in patients with SCD will seek clinical proof of concept for VOC prevention.

The management of factor Xa (FXa) inhibitor-associated bleeding remains a clinical challenge. Massive bleeding is often associated with complex coagulopathy and, thus, the sole reversal of FXa inhibitors might not be sufficient to restore hemostasis, requiring instead a multimodal approach. Four-factor prothrombin complex concentrate (4F-PCC) is widely recognized as a viable treatment option for FXa inhibitor-associated bleeding. Here, we applied computational models to explore the effect 4F-PCC has on the coagulation cascade and restoration of thrombin generation in a system that simulates a patient that has received a FXa inhibitor. The coagulation model is largely based on a previously developed model with modifications incorporated from various other published sources. The model was calibrated and validated using data from a phase 3 clinical trial of vitamin K antagonist reversal with 4F-PCC. Using the parameters and initial conditions determined during the calibration and validation process, the prothrombin time (PT) test simulations predicted a PT of 11.4 seconds. The model successfully simulated the effects of rivaroxaban and apixaban on total thrombin concentration and showed that 4F-PCC increased thrombin generation in the presence of rivaroxaban or apixaban.

Background and Aims: Replacement FIX therapy (rIX) is an effective treatment for hemophilia B even with undetectable levels in the blood 1. However, the mechanistic reason for hemostasis with low plasma levels is not well understood. There is growing evidence that FIX interactions with one or multiple binding partners (BP), may play a significant role in the exposure and hemostatic efficacy of rIX 2,3. The aim of this study is to explore this hypothesis by comparing the plasma PK, tissue biodistribution, and in vivo endpoints of different rIX variants using a mouse QSP model.

Despite intensive efforts in recent years, a curative therapy for cutaneous T-cell lymphoma (CTCL) has not yet been developed. Therefore, the establishment of new therapeutic approaches with higher efficacy rates and milder side effects is strongly desired. A characteristic feature of the malignant T-cell population in CTCL is resistance toward cell death resulting from constitutive NF-κB activation. Therefore, NF-κB-dependent cell death resistance represents an interesting therapeutic target in CTCL because an NF-κB-directed therapy would leave bystander T cells widely unaffected. We investigated the effects of dimethyl fumarate (DMF) on CTCL cells in vitro and in vivo. DMF induced cell death in primary patient-derived CD4(+) cells and CTCL cell lines, but hardly in T cells from healthy donors. DMF-induced cell death was linked specifically to NF-κB inhibition. To study the impact of DMF in vivo, we developed 2 CTCL xenograft mouse models with different cutaneous localizations of the T-cell infiltrate. DMF treatment delayed the growth of CTCL tumors and prevented formation of distant metastases. In addition, DMF induced increased cell death in primary CTCL tumors and in liver metastases. In summary, DMF treatment represents a remarkable therapeutic option in CTCL because it restores CTCL apoptosis in vitro and in preclinical models in vivo and prevents spreading of the disease to distant sites. DMF treatment is of particular promise in CTCL because DMF is already in successful clinical use in the treatment of psoriasis and multiple sclerosis allowing fast translation into clinical studies in CTCL.

NF-κB is an important transcription factor in the immune system, and aberrant NF-κB activity contributes to malignant diseases and autoimmunity. In T cells, NF-κB is activated upon TCR stimulation, and signal transduction to NF-κB activation is triggered by a cascade of phosphorylation events. However, fine-tuning and termination of TCR signaling are only partially understood. Phosphatases oppose the role of kinases by removing phosphate moieties. The catalytic activity of the protein phosphatase PP2A has been implicated in the regulation of NF-κB. PP2A acts in trimeric complexes in which the catalytic subunit is promiscuous and the regulatory subunit confers substrate specificity. To understand and eventually target NF-κB-specific PP2A functions it is essential to define the regulatory PP2A subunit involved. So far, the regulatory PP2A subunit that mediates NF-κB suppression in T cells remained undefined. By performing a siRNA screen in Jurkat T cells harboring a NF-κB-responsive luciferase reporter, we identified the PP2A regulatory subunit B56γ as negative regulator of NF-κB in TCR signaling. B56γ was strongly up-regulated upon primary human T cell activation, and B56γ silencing induced increased IκB kinase (IKK) and IκBα phosphorylation upon TCR stimulation. B56γ silencing enhanced NF-κB activity, resulting in increased NF-κB target gene expression including the T cell cytokine IL-2. In addition, T cell proliferation was increased upon B56γ silencing. These data help to understand the physiology of PP2A function in T cells and the pathophysiology of diseases involving PP2A and NF-κB.

Signal transduction to nuclear factor-kappa B (NF-κB) involves multiple kinases and phosphorylated target proteins, but little is known about signal termination by dephosphorylation. By RNAi screening, we have identified protein phosphatase 4 regulatory subunit 1 (PP4R1) as a negative regulator of NF-κB activity in T lymphocytes. PP4R1 formed part of a distinct PP4 holoenzyme and bridged the inhibitor of NF-κB kinase (IKK) complex and the phosphatase PP4c, thereby directing PP4c activity to dephosphorylate and inactivate the IKK complex. PP4R1 expression was triggered upon activation and proliferation of primary human T lymphocytes and deficiency for PP4R1 caused sustained and increased IKK activity, T cell hyperactivation, and aberrant NF-κB signaling in NF-κB-addicted T cell lymphomas. Collectively, our results unravel PP4R1 as a previously unknown activation-associated negative regulator of IKK activity in lymphocytes whose downregulation promotes oncogenic NF-κB signaling in a subgroup of T cell lymphomas.

S OF LECTURES............................................................................................................................ 1 GENETIC SCREENING FOR PHOSPHATASES THAT REGULATE NF-B ACTIVITY IN T LYMPHOCYTES ................................................................................................................................................. 2 MARKUS BRECHMANN, THOMAS MOCK, DOROTHEE NICKLES, FELICE FREY, MICHAEL BOUTROS, PETER H. KRAMMER, AND RÜDIGER ARNOLD REGULATION OF SPONTANEOUS T CELL RESPONSES IN CANCER PATIENTS ............................ 3

NF-κB is a crucial transcription factor regulating apoptosis sensitivity and resistance. It has been shown that inhibition of NF-κB in T lymphocytes leads to sensitization towards apoptosis. The underlying molecular mechanism is not entirely understood. Therefore, we investigated T cell receptor (TCR) stimulated apoptosis in T cells in which NF-κB activity is blocked by an inhibitor or IκBα overexpression. We show that enhanced apoptosis upon TCR stimulation is caspase- and JNK-dependent, but independent of the CD95/CD95L system. Generation of reactive oxygen species (ROS) induced sustained JNK phosphorylation by inactivation of MAP kinase phosphatase 7 (MKP7). Sustained JNK activation causes upregulation of the pro-apototic protein BIM. Thus, inhibition of NF-κB causes a switch from classical activation-induced cell death (AICD) to CD95L-independent apoptosis.

Activation of the NF-kappaB pathway in T cells is required for induction of an adaptive immune response. Hematopoietic progenitor kinase (HPK1) is an important proximal mediator of T-cell receptor (TCR)-induced NF-kappaB activation. Knock-down of HPK1 abrogates TCR-induced IKKbeta and NF-kappaB activation, whereas active HPK1 leads to increased IKKbeta activity in T cells. Yet, the precise molecular mechanism of this process remains elusive. Here, we show that HPK1-mediated NF-kappaB activation is dependent on the adaptor protein CARMA1. HPK1 interacts with CARMA1 in a TCR stimulation-dependent manner and phosphorylates the linker region of CARMA1. Interestingly, the putative HPK1 phosphorylation sites in CARMA1 are different from known PKC consensus sites. Mutations of residues S549, S551, and S552 in CARMA1 abrogated phosphorylation of a CARMA1-linker construct by HPK1 in vitro. In addition, CARMA1 S551A or S5549A/S551A point mutants failed to restore HPK1-mediated and TCR-mediated NF-kappaB activation and IL-2 expression in CARMA1-deficient T cells. Thus, we identify HPK1 as a kinase specific for CARMA1 and suggest HPK1-mediated phosphorylation of CARMA1 as an additional regulatory mechanism tuning the NF-kappaB response upon TCR stimulation.

The family of SLPs (Src homology 2 domain-containing leukocyte adaptor proteins) are cytoplasmic signal effectors of lymphocyte antigen receptors. A main function of SLP is to orchestrate the assembly of Ca2+-mobilizing enzymes at the inner leaflet of the plasma membrane. For this purpose, SLP-76 in T cells utilizes the transmembrane adaptor LAT, but the mechanism of SLP-65 membrane anchoring in B cells remains an enigma. We now employed two genetic reconstitution systems to unravel structural requirements of SLP-65 for the initiation of Ca2+ mobilization and subsequent activation of gene transcription. First, mutational analysis of SLP-65 in DT40 B cells revealed that its C-terminal Src homology 2 domain controls efficient tyrosine phosphorylation by the kinase Syk, plasma membrane recruitment, as well as downstream signaling to NFAT activation. Second, we dissected these processes by expressing SLP-65 in SLP-76-deficient T cells and found that a kinase-independent adaptor function of Syk is required to link phosphorylated SLP-65 to Ca2+ mobilization. These approaches unmask a mechanistic complexity of SLP-65 activation and coupling to signaling cascades in that Syk is upstream as well as downstream of SLP-65. Moreover, membrane anchoring of the SLP-65-assembled Ca2+ initiation complex, which appears to be fundamentally different from that of closely related SLP-76, does not necessarily involve a B cell-specific component.

Mice with combined deficiencies of the low-density lipoprotein receptor (LDLR(-/-)) and the catalytic component of an apolipoprotein B-edisome complex (APOBEC1(-/-)) that converts apoB-100 to apoB-48 have been characterized, and this model of LDL cholesterol-driven atherosclerosis was applied to an investigation of the role of fibrinogen (Fg) in the genesis and progression of the plaque. LDLR(-/-)/APOBEC1(-/-)/FG(-/-) (L(-/-)/A(-/-)/FG(-/-)) triple-deficient mice presented more advanced plaque in their aortic trees and aortic sinuses at 24, 36, and 48 weeks of age compared to L(-/-)/A(-/-) mice, a feature that may result from enhanced platelet activation in these former mice. This is supported by the presence of hypercoagulability, increased CD61 and CD62P on resting platelets, and higher plasma soluble P-selectin in L(-/-)/A(-/-)/FG(-/-) mice as compared to L(-/-)/A(-/-), FG(-/-), or wild-type mice. The elevated higher molecular weight forms of von Willebrand factor (VWF) in L(-/-)/A(-/-)/FG(-/-) mice, revealed by increased VWF collagen binding activity, perhaps resulting from down-regulation of its cleaving metalloproteinase, ADAMTS13, further indicates enhanced platelet activation. Thus, the earlier arterial plaque deposition in L(-/-)/A(-/-)/FG(-/-) mice appears to contain a contribution from enhanced levels of thrombin and activated platelets, a synergistic consequence of an Fg deficiency combined with a high LDL cholesterol concentration.