Secretion of von Willebrand Factor and Suppression of ADAMTS-13 Activity by Markedly High Concentration of Ferritin.

Hyperferritinemia is associated with poor outcomes in critically ill patients with sepsis, hemophagocytic lymphohistiocytosis (HLH), macrophage activation syndromes (MAS) and coronavirus disease 19 (COVID-19). Autopsies of hyperferritinemic patients that succumbed to either sepsis, HLH, MAS or COVID-19 have revealed disseminated microvascular thromboses with von Willebrand factor (VWF)-, platelets-, and/or fibrin-rich microthrombi. It is unknown whether high plasma ferritin concentration actively promotes microvascular thrombosis, or merely serves as a prognostic biomarker in these patients. Here, we show that secretion of VWF from human umbilical vein endothelial cells (HUVEC) is significantly enhanced by 100,000 ng/ml of recombinant ferritin heavy chain protein (FHC). Ferritin fraction that was isolated by size exclusion chromatography from the plasma of critically ill HLH patients promoted VWF secretion from HUVEC, compared to similar fraction from non-critically ill control plasma. Furthermore, recombinant FHC moderately suppressed the activity of VWF cleaving metalloprotease ADAMTS-13. These observations suggest that a state of marked hyperferritinemia could promote thrombosis and organ injury by inducing endothelial VWF secretion and reducing the ADAMTS-13 activity.

A monoclonal antibody with broad specificity for the ligands of insulin B:9-23 reactive T cells prevents spontaneous type 1 diabetes in mice.

Activation of T cells specific for insulin B chain amino acids 9 to 23 (B:9-23) is essential for the initiation of type 1 diabetes (T1D) in non-obese diabetic mice. We previously reported that peptide/MHC complexes containing optimized B:9-23 mimotopes can activate most insulin-reactive pathogenic T cells. A monoclonal antibody (mAb287) targeting these complexes prevented disease in 30-50% of treated animals (compared to 10% of animals given an isotype control). The incomplete protection is likely due to the relatively low affinity of the antibody for its ligand and limited specificity. Here, we report an enhanced reagent, mAb757, with improved specificity, affinity, and efficacy in modulating T1D. Importantly, mAb757 bound with nanomolar affinity to agonists of both "type A" and "type B" cells and suppressed "type B" cells more efficiently than mAb287. When given weekly starting at 4 weeks of age, mAb757 protected ~70% of treated mice from developing T1D for at least 35 weeks, while mAb287 only delayed disease in 25% of animals under the same conditions. Consistent with its higher affinity, mAb757 was also able to stain antigen-presenting cells loaded with B:9-23 mimotopes in vivo. We conclude that monoclonal antibodies that can block the presentation of pathogenic T cell receptor epitopes are viable candidates for antigen-specific immunotherapy for T1D.

Modulating the rate of fibrin formation and clot structure attenuates microvascular thrombosis in systemic inflammation.

Systemic inflammation can lead to coagulopathy and disseminated intravascular coagulation (DIC). In prior studies, the recombinant A2 domain of human von Willebrand factor (VWF; A2 protein) attenuated DIC and decreased mortality in lipopolysaccharide (LPS)-treated mice. Here, we performed studies to dissect the mechanism by which the A2 protein moderates DIC. We used confocal microscopy to analyze the fibrin clot structure in plasma from healthy humans and endotoxemic mice, turbidity assays to examine fibrin polymerization, and a murine model for LPS-induced DIC and introduced a loss-of-function mutation into the A2 protein for fibrin. The mutation of the residue E1567 located in the α2 helix of the folded A2 domain of VWF inhibited binding activity for fibrin, possibly mapping a novel region containing a putative binding site for fibrin. The A2 protein increased the initial rate of change of fibrin polymerization, intercalated into the fibrin network, and modified the resultant clot structure in vitro. Furthermore, ex vivo experiments using plasma from mice with endotoxemia treated with the A2 protein revealed an increased rate of fibrin formation and an altered clot structure as compared with plasma from nontreated sick animals. Moreover, and in contrast to the A2 mutant, the A2 protein improved survival and reduced fibrin deposition and microvascular thrombosis in mice with endotoxemia-induced DIC. Importantly, in vivo and in vitro studies indicated that the A2 protein did not affect experimental thrombosis. Thus, we provide evidence for a novel treatment to attenuate systemic inflammation-induced coagulopathy/DIC via targeting fibrin formation, without an increased risk for bleeding.

High-Efficiency Generation of Antigen-Specific Primary Mouse Cytotoxic T Cells for Functional Testing in an Autoimmune Diabetes Model.

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the spontaneous non-obese diabetes (NOD) mouse model, insulin is the primary target, and genetic manipulation of these animals to remove a single key insulin epitope prevents disease. Thus, selective elimination of professional antigen presenting cells (APCs) bearing this pathogenic epitope is an approach to inhibit the unwanted insulin-specific autoimmune responses, and likely has greater translational potential. Chimeric antigen receptors (CARs) can redirect T cells to selectively target disease-causing antigens. This technique is fundamental to recent attempts to use cellular engineering for adoptive cell therapy to treat multiple cancers. In this protocol, we describe an optimized T-cell retrovirus (RV) transduction and in vitro expansion protocol that generates high numbers of functional antigen-specific CD8 CAR-T cells starting from a low number of naive cells. Previously multiple CAR-T cell protocols have been described, but typically with relatively low transduction efficiency and cell viability following transduction. In contrast, our protocol provides up to 90% transduction efficiency, and the cells generated can survive more than two weeks in vivo and significantly delay disease onset following a single infusion. We provide a detailed description of the cell maintenance and transduction protocol, so that the critical steps can be easily followed. The whole procedure from primary cell isolation to CAR expression can be performed within 14 days. The general method may be applied to any mouse disease model in which the target is known. Similarly, the specific application (targeting a pathogenic peptide/MHC class II complex) is applicable to any other autoimmune disease model for which a key complex has been identified.

Chimeric antigen receptor (CAR) T cells targeting a pathogenic MHC class II:peptide complex modulate the progression of autoimmune diabetes.

A primary initiating epitope in the NOD mouse model of Type 1 Diabetes (T1D) lies between residues 9 and 23 of the insulin B chain. The B:9-23 peptide can bind to the NOD MHC class II molecule (I-Ag7) in multiple registers, but only one, (register 3, R3), creates complexes able to stimulate the majority of pathogenic B:9-23-specific CD4+ T cells. Previously we generated a monoclonal antibody (mAb287) that targets this critical I-Ag7-B:9-23(R3) complex. When given weekly to pre-diabetic mice at either early or late stages of disease, mAb287 was able to delay or prevent T1D in the treated animals. Although the precise mechanism of action of mAb287 remains unclear, we hypothesized that it may involve deletion of antigen presenting cells (APCs) bearing the pathogenic IAg7-B:9-23(R3) complexes, and that this process might be rendered more efficient by re-directing cytotoxic T cells using a mAb287 chimeric antigen receptor (287-CAR). As anticipated, 287-CAR T cells secreted IFN-γ in response to stimulation by I-Ag7-B:9-23(R3) complexes expressed on artificial APCs, but not I-Ag7 loaded with other peptides, and killed the presenting cells in vitro. A single infusion of 287-CAR CD8+ T cells to young (5 week old) NOD mice significantly delayed the onset of overt hyperglycemia compared to untreated animals (p = 0.022). None of the 287-CAR CD8+ T cell treated mice developed diabetes before 18 weeks of age, while 29% of control-CAR T cell treated mice (p = 0.044) and 52% of the un-treated mice (p = 0.0001) had developed T1D by this time. However, the protection provided by 287-CAR CD8+ T cells declined with time, and no significant difference in overall incidence by 30 weeks between the 3 groups was observed. Mechanistic studies indicated that the adoptively transferred 287-CAR T cells selectively homed to pancreatic lymph nodes, and in some animals could persist for at least 1-2 weeks post-transfer, but were essentially undetectable 10-15 weeks later. Our study demonstrates that CAR T cells specific for a pathogenic MHC class II:peptide complex can be effective in vivo, but that a single infusion of the current iteration can only delay, but not prevent, the development of T1D. Future studies should therefore be directed towards optimizing strategies designed to improve the longevity of the transferred cells.