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Additional file 1: Fig. S1. Assessment of anti-PD-1 scFv binding. (A) Transgene schematic of bicistronic vector for constitutive secretion of anti-PD-1 scFv; (B) Supernatant from transfected HEK-293T cells producing anti-PD-1scFv was tested to confirm binding to PD-1+SupT1 cell line via flow cytometry; (C) Binding affinity measured via SPR using an anti-PD-1 scFv-Fc coated CM5 Chip: representative sensogram (of 3 independent repeats) of response units (RU) to increasing concentrations of PD-1 protein (0µg/ml–2.5µg/ml). Fig. S2. Transduction efficiency and additional in vitro characterization of F CAR and F i-CAR-T cells effector functions. (A) Representative flow cytometry plots of F CAR and F i-CAR T cells demonstrating comparable transduction levels between constructs as assessed by mCherry expression; (B) Representative flow cytometry of ROR1 and PD-L1 expression on positive MDA-MB-231, H1975 and negative control SUPT1 cell lines; (C) Anti-PD1 scFv was Luciferase-tagged and co-culture conditions were tested for scFv production via addition of substrate (coelenterazine) and luminescence quantified. Results are mean from 3 donors in triplicate; (D) Cytotoxicity assay following 72h of co-culture with CAR-T cells, viable ROR1- target cell lines were assessed via flow cytometry, results are mean+SD of 3 donors in triplicates normalized to control CD19 CAR-T cells; (E) Immune checkpoint expression was assessed via flow cytometry and shown as % of CAR-T cells. 2way ANOVA used: *p

Thrombus formation leading to vaso-occlusive events is a major cause of death, and involves complex interactions between coagulation, fibrinolytic and innate immune systems. Leukocyte recruitment is a key step, mediated partly by chemotactic complement activation factors C3a and C5a. However, mechanisms mediating C3a/C5a generation during thrombosis have not been studied. In a murine venous thrombosis model, levels of thrombin–antithrombin complexes poorly correlated with C3a and C5a, excluding a central role for thrombin in C3a/C5a production. However, clotweight strongly correlated with C5a, suggesting processes triggered during thrombosis promote C5a generation. Since thrombosis elicits fibrinolysis,we hypothesized that plasmin activates C5 during thrombosis. In vitro, the catalytic efficiency of plasmin-mediated C5a generation greatly exceeded that of thrombin or factor Xa, but was similar to the recognized complement C5 convertases. Plasmin-activated C5 yielded a functional membrane attack complex (MAC). In an arterial thrombosis model, plasminogen activator administration increased C5a levels. Overall, these findings suggest plasmin bridges thrombosis and the immune response by liberating C5a and inducing MAC assembly. These new insights may lead to the development of strategies to limit thrombus formation and/or enhance resolution

Thrombus formation leading to vaso-occlusive events is a major cause of death, and involves complex interactions between coagulation, fibrinolytic and innate immune systems. Leukocyte recruitment is a key step, mediated partly by chemotactic complement activation factors C3a and C5a. However, mechanisms mediating C3a/C5a generation during thrombosis have not been studied. In a murine venous thrombosis model, levels of thrombin–antithrombin complexes poorly correlated with C3a and C5a, excluding a central role for thrombin in C3a/C5a production. However, clot weight strongly correlated with C5a, suggesting processes triggered during thrombosis promote C5a generation. Since thrombosis elicits fibrinolysis, we hypothesized that plasmin activates C5 during thrombosis. In vitro, the catalytic efficiency of plasmin-mediated C5a generation greatly exceeded that of thrombin or factor Xa, but was similar to the recognized complement C5 convertases. Plasmin-activated C5 yielded a functional membrane attack complex (MAC). In an arterial thrombosis model, plasminogen activator administration increased C5a levels. Overall, these findings suggest plasmin bridges thrombosis and the immune response by liberating C5a and inducing MAC assembly. These new insights may lead to the development of strategies to limit thrombus formation and/or enhance resolution.