Human Fc gamma receptor IIIA blockade inhibits platelet destruction in a humanized murine model of ITP.

ABSTRACT: Fc gamma receptor (FcγR) IIIA is an important receptor for immunoglobulin G (IgG) and is involved in immune defense mechanisms as well as tissue destruction in some autoimmune diseases including immune thrombocytopenia (ITP). FcγRIIIA on macrophages can trigger phagocytosis of IgG-sensitized platelets, and prior pilot studies observed blockade of FcγRIIIA increased platelet counts in patients with ITP. Unfortunately, although blockade of FcγRIIIA in patients with ITP increased platelet counts, its engagement by the blocking antibody drove serious adverse inflammatory reactions. These adverse events were postulated to originate from the antibody's Fc and/or bivalent nature. The blockade of human FcγRIIIA in vivo with a monovalent construct lacking an active Fc region has not yet been achieved. To effectively block FcγRIIIA in vivo, we developed a high affinity monovalent single-chain variable fragment (scFv) that can bind and block human FcγRIIIA. This scFv (17C02) was expressed in 3 formats: a monovalent fusion protein with albumin, a 1-armed human IgG1 antibody, and a standard bivalent mouse (IgG2a) antibody. Both monovalent formats were effective in preventing phagocytosis of ITP serum-sensitized human platelets. In vivo studies using FcγR-humanized mice demonstrated that both monovalent therapeutics were also able to increase platelet counts. The monovalent albumin fusion protein did not have adverse event activity as assessed by changes in body temperature, whereas the 1-armed antibody induced some changes in body temperature even though the Fc region function was impaired by the Leu234Ala and Leu235Ala mutations. These data demonstrate that monovalent blockade of human FcγRIIIA in vivo can potentially be a therapeutic strategy for patients with ITP.

Serum from half of patients with immune thrombocytopenia trigger macrophage phagocytosis of platelets.

Humoral antiplatelet factors, such as autoantibodies, are thought to primarily clear platelets by triggering macrophage phagocytosis in immune thrombocytopenia (ITP). However, there are few studies characterizing the capacity and mechanisms of humoral factor-triggered macrophage phagocytosis of platelets using specimens from patients with ITP. Here, we assessed sera from a cohort of 24 patients with ITP for the capacity to trigger macrophage phagocytosis of normal donor platelets and characterized the contribution of humoral factors to phagocytosis. Sera that produced a phagocytosis magnitude greater than a normal human serum mean + 2 standard deviations were considered phagocytosis-positive. Overall, 42% (8/19) of MHC I alloantibody-negative ITP sera were phagocytosis-positive. The indirect monoclonal antibody immobilization of platelet antigens assay was used to detect immunoglobulin G (IgG) autoantibodies to glycoproteins (GP)IIb/IIIa, GPIb/IX, and GPIa/IIa. Autoantibody-positive sera triggered a higher mean magnitude of phagocytosis than autoantibody-negative sera. Phagocytosis correlated inversely with platelet counts among autoantibody-positive patients but not among autoantibody-negative patients. Select phagocytosis-positive sera were separated into IgG-purified and -depleted fractions via protein G and reassessed for phagocytosis. Phagocytosis was largely retained in the purified IgG fractions. In addition, we assessed serum concentrations of C-reactive protein, serum amyloid P, and pentraxin 3 as potential phagocytosis modulators. Pentraxin 3 concentrations correlated inversely with platelet counts among patients positive for autoantibodies. Taken together, sera from approximately half of the patients with ITP studied triggered macrophage phagocytosis of platelets beyond a normal level. An important role for antiplatelet autoantibodies in phagocytosis is supported; a role for pentraxins such as pentraxin 3 may be suggested.

THP-1 cells transduced with CD16A utilize Fcγ receptor I and III in the phagocytosis of IgG-sensitized human erythrocytes and platelets.

Fc gamma receptors (FcγRs) are critical effector receptors for immunoglobulin G (IgG) antibodies. On macrophages, FcγRs mediate multiple effector functions, including phagocytosis, but the individual contribution of specific FcγRs to phagocytosis has not been fully characterized. Primary human macrophage populations, such as splenic macrophages, can express FcγRI, FcγRIIA, and FcγRIIIA. However, there is currently no widely available monocyte or macrophage cell line expressing all these receptors. Common sources of monocytes for differentiation into macrophages, such as human peripheral blood monocytes and the monocytic leukemia cell line THP-1, generally lack the expression of FcγRIIIA (CD16A). Here, we utilized a lentiviral system to generate THP-1 cells stably expressing human FcγRIIIA (CD16F158). THP-1-CD16A cells treated with phorbol 12-myristate 13-acetate for 24 hours phagocytosed anti-D-opsonized human red blood cells primarily utilizing FcγRI with a lesser but significant contribution of IIIA while phagocytosis of antibody-opsonized human platelets equally utilized FcγRI and Fcγ IIIA. Despite the well-known ability of FcγRIIA to bind IgG in cell free systems, this receptor did not appear to be involved in either RBC or platelet phagocytosis. These transgenic cells may constitute a valuable tool for studying macrophage FcγR utilization and function.

Comparison of methods for pre-processing, exosome isolation, and RNA extraction in unpasteurized bovine and human milk.

Milk is a highly complex, heterogeneous biological fluid that contains non-nutritive, bioactive extracellular vesicles called exosomes. Characterization of milk-derived exosomes (MDEs) is challenging due to the lack of standardized methods that are currently being used for milk pre-processing, storage, and exosome isolation. In this study, we tested: 1) three pre-processing methods to remove cream, fat, cellular debris, and casein proteins from bovine milk to determine whether pre-processing of whole milk prior to long-term storage improves MDE isolations, 2) the suitability of two standard exosome isolation methods for MDE fractionation, and 3) four extraction protocols for obtaining high quality RNA from bovine and human MDEs. MDEs were characterized via Transmission Electron Microscopy (TEM), Nanoparticle Tracking Analysis (NTA), and western immunoblotting for CD9, CD63, and Calnexin protein markers. We also present an optimized method of TEM sample preparation for MDEs. Our results indicate that: 1) Removal of cream and fat globules from unpasteurized bovine milk, prior to long-term storage, improves the MDE yield but not purity, 2) Differential ultracentrifugation (DUC) combined with serial filtration is better suited for bovine MDE isolation compared to ExoQuick (EQ) combined with serial filtration, however both methods were comparable for human milk, and 3) TRIzol LS is better suited for RNA extraction from bovine MDEs isolated by EQ and DUC methods. 4) TRIzol LS, TRIzol+RNA Clean and Concentrator, and TRIzol LS+RNA Clean and Concentrator methods can be used for RNA extractions from human MDEs isolated by EQ, yet the TRIzol LS method is better suited for human MDEs isolated by DUC. The QIAzol + miRNeasy Mini Kit produced the lowest RNA yield for bovine and human MDEs.