CHARACTERIZING EPITOPES AND ISOTYPES OF CROHN'S DISEASE-ASSOCIATED ANTI-GRANULOCYTE MACROPHAGE-COLONY STIMULATING FACTOR ANTIBODIES.

Background
Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) maintains mononuclear phagocytes (MNP) and supports intestinal immune homeostasis. Anti-GM-CSF autoantibodies (aGMAbs) are found in approx. 25% of all Crohn’s disease (CD) patients. They precede the diagnosis of CD by six years and associate with complicated disease at the time of diagnosis. Anti-GMAbs neutralize GM-CSF by binding to post-translational glycosylations, resulting in altered GM-CSF receptor signaling through STAT5 in MNP. However, the isotype profile and epitopes of aGMAbs in CD remain unknown. Identifying the epitope/s and characterizing the isotype profile of aGMAbs in CD patients could advance our understanding of CD pathogenesis and inform about changes in aGMAb reactivity and isotype profile prior to diagnosis and after diagnosis.

Methods
To characterize aGMAbs, we developed a bead-based multiplex flow cytometric assay allowing the identification of glyco-epitopes and the accompanying isotype profiles. We generated genetically engineered GM-CSF variants lacking all, or individual glycosylation sites and employ a multicolor anti-isotype analysis to determine the epitope/s of aGMAbs. The developed beads are further useful to assess GM-CSF signaling in CD patients with aGMAbs.

Results
CD patient sera containing aGMAbs bind to glycosylated human recombinant GM-CSF but not genetically engineered human GM-CSF lacking all glycosylation sites, when the cytokine is coupled to fluorescent beads. Binding of CD-associated aGMAbs to GM-CSF on fluorescent beads is blocked by pre-incubation of aGMAb-containing CD serum with fully glycosylated GM-CSF (Fig.1A). Genetically engineered GM-CSF glycovariants can be efficiently conjugated to fluorescent beads (Fig.1B). Multiplexing 8 beads of different diameters (0.2µm – 2µm) and two fluorescent dyes (green and blue), each engineered GM-CSF glycovariant can be resolved in the pool of 8 beads by multicolor flow cytometry (Fig.1C). Analysis of the isotype profile on fully glycosylated GM-CSF variants revealed a 58.4±60.4, 10.5±14.1 and 11.9±17.2-fold higher mean fluorescent intensity (MFI) for IgM, IgG and IgA aGMAbs in CD patient sera (n=10) respectively, when compared to controls. Moreover, the engineered GM-CSF glycovariants retain their ability to engage the GM-CSF receptor on MNP, even after coupling to fluorescent beads (Fig.1D). This demonstrates the suitability of this assay to identify aGMAb epitope/s, characterize their isotype profile and their ability to impair GM-CSF signaling on MNP using minimal amounts of patient serum (5µl) (Fig.1D).

Conclusions
We report a new bead-based technology for commercial flow cytometers, allowing the epitope and isotype characterization of CD-associated aGMAbs. The developed assay further allows functional characterization of MNP and will help to better define patients subsets.