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Identifying epitopes of anti-FcaRI monoclonal antibodies on FcaRI ectodomain that trigger the anti-inflammatory ITAMi signaling pathway

Parthasarathy, Upasana
http://rave.ohiolink.edu/etdc/view?acc_num=ucin1418910350

Abstract Details

2014, MS, University of Cincinnati, Medicine: Immunology.
Autoimmune disorders are the second leading type of chronic illness in the US, affecting about 50 million individuals. In 2001, the National Institute of Allergy and Infectious Diseases (NIAID) estimated that the annual autoimmune healthcare cost is greater than $100 billion, although this value may be significantly underestimated. Autoimmune diseases such as hypersensitivity reactions are a result of impairment of this FcR regulatory system. Here, we focus on the interaction between Immunoglobulin A (IgA), the most predominant antibody in mucosal sites, and its principal myeloid receptor, FcaRI (CD89). Cross-linking of multiple FcaRI molecules at the cell surface by immune complexed IgA, initiates pro-inflammatory immune responses such as phagocytosis, antigen presentation and antibody-dependent cellular cytotoxicity. These findings led to perceiving FcaRI as a solely activating receptor. In 2005, Pasquier et al. identified that FcaRI, when interacting with monomeric serum IgA, can also drive powerful anti-inflammatory responses through the ITAMs in the FcR γ-chain and thus behave as an inhibitory receptor that controls inflammation. This revealed the dual nature of the ITAM motif, which otherwise is typically considered to be involved in immune cell activation. This inhibitory signaling pathway mediated by FcaRI in association with FcRγ ITAMs was termed the Inhibitory ITAM or ITAMi signaling pathway. Several research groups have shown that some but not all anti-FcaRI monoclonal antibody Fab fragments are capable of triggering the ITAMi pathway via FcaRI. The anti-FcaRI monoclonal antibodies most widely used to study the ITAMi pathway include MIP8a, A3, A59 etc. and these antibodies recognize different extracellular domains in the FcaRI ectodomain. Hence FcaRI can be considered as a 3 state system: a resting state in which it does not mediate signaling, an activating state in which it triggers pro-inflammatory responses via recruitment of a Syk kinase, and an inhibiting state in which it triggers anti-inflammatory responses via recruitment of SHP-1 phosphatase. Based on this, we hypothesize that anti-FcaRI monoclonal antibodies which monovalently target FcaRI and trigger the ITAMi pathway have their antigenic epitopes clustered in certain regions of the ectodomain, forming hotspots. Identifying key amino acid residues or sequences of residues in these hotspots will enable recognition of optimal regions in FcaRI ectodomain that can be targeted to trigger the ITAMi pathway. We aim to lay the groundwork to define the characteristics of ligands capable of triggering ITAMi signaling via FcaRI and perform experiments to determine how different FcaRI binding ligands are able to trigger two contrasting pathways solely through the ITAM of the accessory FcRγ receptor. Therefore, the main objective of this thesis is to identify the antigenic epitope of anti-FcaRI monoclonal antibody A59 on FcaRI ectodomain using site-directed mutagenesis and enzyme linked immunosorbent assays. Understanding the mechanistic basis for ITAMi signaling is a necessary step towards effectively triggering ITAMi responses in immune cells by targeting FcaRI. We believe that this should aid in better defining the characteristics of ligands that are capable of triggering the inhibitory function of FcaRI and characterizing the clustering mode of FcaRI in the three functional states.
Andrew Herr, Ph.D. (Committee Chair)
Jonathan Katz, Ph.D. (Committee Member)
William Miller, Ph.D. (Committee Member)
78 p.
Immunology
FcalphaRI ectodomain; antigenic epitopes; A59; ITAMi; epitope mapping; WT FcalphaRI EC- Fcgamma fusion protein

Recommended Citations

Citations

  • Parthasarathy, U. (2014). Identifying epitopes of anti-FcaRI monoclonal antibodies on FcaRI ectodomain that trigger the anti-inflammatory ITAMi signaling pathway [Master's thesis, University of Cincinnati]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1418910350

    APA Style (7th edition)

  • Parthasarathy, Upasana. Identifying epitopes of anti-FcaRI monoclonal antibodies on FcaRI ectodomain that trigger the anti-inflammatory ITAMi signaling pathway. 2014. University of Cincinnati, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ucin1418910350.

    MLA Style (8th edition)

  • Parthasarathy, Upasana. "Identifying epitopes of anti-FcaRI monoclonal antibodies on FcaRI ectodomain that trigger the anti-inflammatory ITAMi signaling pathway." Master's thesis, University of Cincinnati, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1418910350

    Chicago Manual of Style (17th edition)

ucin1418910350
576
© 2014, some rights reserved.Creative Commons License Identifying epitopes of anti-FcaRI monoclonal antibodies on FcaRI ectodomain that trigger the anti-inflammatory ITAMi signaling pathway by Upasana Parthasarathy is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. Based on a work at etd.ohiolink.edu.
This open access ETD is published by University of Cincinnati and OhioLINK.