Functional significance of the interaction between inducible costimulator (ICOS) and its ligand (ICOSL)
MetadataShow full item record
BACKGROUND Inducible costimulator (ICOS) and its ligand (ICOSL) are a pair of costimulatory molecules that co-localize in germinal centers (GC). This interaction is critical for the maturation of GC B cells to affinity-matured memory B cells and long-lived plasma cells. Both ICOS and ICOSL are implicated in systemic lupus erythematosus (SLE). It is known that ICOSL sheds from the cell membrane and that the soluble form of ICOSL (sICOSL) is elevated in SLE; though the function of sICOSL is poorly understood. While it is known that binding of ICOSL on antigen-presenting cells (APC) to ICOS on T cells leads to cell signaling resulting in T cell activation and differentiation, there is also some preliminary evidence that reverse signaling may also occur through ICOSL in APCs. The binding interaction between ICOS and sICOSL has not been fully characterized and is important to understand if either molecule is to be targeted therapeutically. The hypothesis evaluated in this study was that the ICOS: ICOSL interaction is a potent and critical mediator of proinflammatory signaling and immune activation that functions both via activated T cell-mediated forward signaling and APC-mediated reverse signaling mechanisms and that ectodomain shedding of ICOSL is a protective mechanism that leads to down-regulation of the proinflammatorysignaling cascade initiated by this interaction. The aim of this thesis is to characterize the binding interaction between ICOS and ICOSL and to provide a review of the literature and discuss future work that would enhance the biological understanding of this interaction and its role in lupus and other autoimmune diseases. METHODS The binding interaction between ICOS and ICOSL was characterized using both soluble proteins and cells with expressed recombinant proteins. Purified soluble ICOSL (sICOSL) was characterized using size-exclusion chromatography multiangle light scattering (SEC-MALS). Surface plasmon resonance (SPR) was used to measure the binding affinity between sICOSL and human ICOS fused to the fragment crystallizable (Fc) portion of an immunoglobulin molecule (hICOS.Fc). The binding interaction was further characterized to account for avidity between hICOS.Fc and sICOSL and between hICOS.Fc and ICOSL expressed recombinant on the cell surface using a solution-based binding method. RESULTS Expressed recombinant and purified sICOSL dimerized over time and with increasing temperatures. The sICOSL: hICOS.Fc interaction did not follow a typical 1:1 binding interaction. In-solution binding experiments resulted in a tighter equilibrium dissociation binding constant (KD) than the surface-based results obtained by SPR. The KD for hICOS.Fc binding to human ICOSL(hICOSL) expressed on cells agreed well with the KD for hICOS.Fc to the soluble protein, indicating that the in-solution binding measurement may measure binding avidity rather than affinity and that this may be the more physiologically relevant interaction. CONCLUSIONS I show in the experimental part of this study that the interaction between ICOS and ICOSL is quite potent and that much of the binding strength is due to avidity, or the combined strength of multiple parts of the molecules interacting with one another, rather than the affinity alone. As this interaction is implicated in SLE pathogenesis, it would be useful to develop a clearer understanding of the most relevant physiological form of these molecules (soluble or transmembrane) and of the biological signaling events that are initiated via this interaction in order to determine whether targeting ICOS or ICOSL may be therapeutically viable approaches.