To investigate in vitro the innate immune response to accelerated stress-induced aggregates of intravenous immunoglobulin (IGIV) using a well-defined human cell-line model, and to correlate the innate response to physical properties of the aggregates.
IGIV aggregates were prepared by applying various accelerated stress methods, and particle size, count and structure were characterized. Immune cell activation as tracked by inflammatory cytokines released in response to aggregates was evaluated in vitro using peripheral blood mononuclear cells (PBMC), primary monocytes and immortalized human monocyte-like cell lines.
IGIV aggregates produced by mechanical stress induced higher cytokine release by PBMC and primary monocytes than aggregates formed by other stresses. Results with the monocytic cell line THP-1 paralleled trends in PBMC and primary monocytes. Effects were dose-dependent, enhanced by complement opsonization, and partially inhibited by blocking toll-like receptors (TLR2 and TLR4) and to a lesser extent by blocking Fc gamma receptors (FcγRs).
Stress-induced IGIV aggregates stimulate a dose-dependent cytokine response in human monocytes and THP-1 cells, mediated in part by TLRs, FcγRs and complement opsonization. THP-1 cells resemble primary monocytes in many respects with regard to tracking the innate response to IgG aggregates. Accordingly, the measurement of inflammatory cytokines released by THP-1 cells provides a readily accessible assay system to screen for the potential innate immunogenicity of IgG aggregates. The results also highlight the role of aggregate structure in interacting with the different receptors mediating innate immunity.
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Antigen presenting cell
Cytometric bead array
Fc gamma receptor
High performance liquid chromatography
Nanoparticle tracking analysis
Pathogen associated molecular patterns
Peripheral blood mononuclear cell
Resonant mass measurement
Size exclusion chromatography
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ACKNOWLEDGMENTS AND DISCLOSURES
The authors gratefully thank Daniela Verthelyi and Jennifer Reed for critically reading the manuscript and providing valuable suggestions and comments. The authors are also thankful to Nancy Eller for useful discussions and to Eunbi Cho for assisting with cell-based assays. This work was funded by FDA contract HHSF223201310233C (PI: E. M. Topp). This project was supported in part by an appointment (JK) to the Research Participation Program at the Center for Biologics Evaluation and Research administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and the U.S. Food and Drug Administration. The authors have no conflicts of interest to declare.
Our contributions are an informal communication and represent our own best judgment. These comments do not bind or obligate FDA.
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SDS-PAGE of different aggregated IGIV samples. (a) Non-reduced conditions. (b) Reduced conditions. M, protein marker; UN, unstressed IGIV; HT, heated IGIV; OX, oxidized IGIV; SH, shaken IGIV; ST, stirred IGIV. (JPG 124 kb)
Dose-response of primary monocytes to stirring-induced IGIV aggregates. (a-e) individual cytokines response to aggregates in primary monocytes. Bars represent 1 standard deviation of the mean response of the donors (n = 4). Each colored closed circle represents the average response of an individual donor (n = 5). SI, stimulation index over the response to unstressed IGIV. The x-axis represents the number of micro particles as determined by flow imaging per one million cells. (JPG 705 kb)
Dose-response of THP-1 cells to stirring-induced IGIV aggregates. (a-e) individual cytokine response to aggregates in THP-1 cells. Bars represent 1 standard deviation of the mean response of cell batches (n = 3). Each colored closed circle represents the average response of a different THP-1 cell batch. SI, stimulation index over the response to unstressed IGIV. The x-axis represents the number of particles as determined by flow imaging per one million cells. (JPG 665 kb)
Expression of innate immunity receptors on the surface of THP-1 cells. x-axes represent the fluorescence intensity measured by flow cytometry using the appropriate channel for the fluorescent dye on each antibody, and y-axes represent the counts of labeled cells. Red curves represent unlabeled controls, whereas blue curves represent labeled cells. (JPG 98 kb)
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Moussa, E.M., Kotarek, J., Blum, J.S. et al. Physical Characterization and Innate Immunogenicity of Aggregated Intravenous Immunoglobulin (IGIV) in an In Vitro Cell-Based Model. Pharm Res 33, 1736–1751 (2016). https://doi.org/10.1007/s11095-016-1914-4