Fluorosomes: a convenient new reagent to detect and block multivalent and complex receptor‐ligand interactions

Abstract

We describe for the first time fluorescent virus-like particles decorated with biologically active mono- and multisubunit immune receptors of choice and the basic application of such fluorosomes (FSs) to visualize and target immune receptor-ligand interactions. For that purpose, human embryonic kidney (HEK)-293 cells were stably transfected with Moloney murine leukemia virus (MoMLV) matrix protein (MA) GFP fusion constructs. To produce FSs, interleukins (ILs), IL-receptors (IL-Rs), and costimulatory molecules were fused to the glycosyl phosphatidyl inositol anchor acceptor sequence of CD16b and coexpressed along with MoMLV group-specific antigen-polymerase (gag-pol) in MA::GFP(+) HEK-293 cells. We show that IL-2 decorated but not control-decorated FSs specifically identify normal and malignant IL-2 receptor-positive (IL-2R(+)) lymphocytes by flow cytometry. In addition to cytokines and costimulatory molecules, FSs were also successfully decorated with the heterotrimeric IL-2Rs, allowing identification of IL-2(+) target cells. Specificity of binding was proven by complete inhibition with nonlabeled, soluble ligands. Moreover, IL-2R FSs efficiently neutralized soluble IL-2 and thus induced unresponsiveness of T cells receiving full activation stimuli via T-cell antigen receptor and CD28. FSs are technically simple, multivalent tools for assessing and blocking mono- and multisubunit immune receptor-ligand interactions with natural constituents in a plasma membrane context.

Lipid Rafts, Pseudotyping, and Virus-Like Particles: Relevance of a Novel, Configurable, and Modular Antigen-Presenting Platform

“…In our studies, IL-2 receptor-decorated VLP not only bound but also neutralized soluble IL-2 [136]. IL-2 receptor-decorated VLP thereby displayed cytokine deprivation capabilities commonly associated with regulatory cells [136]. This may open new avenues to exploit ‘regulatory’ VLP for therapeutic purposes.…”

“…These findings seem to be of special interest since CD25+ regulatory CD4+ T cell (nTreg) subsets have been shown to exert their regulatory function on activated effector CD4+ T cells by – among other mechanisms – binding and neutralizing secreted IL-2 and other common γ-chain-(CD132)-associated cytokines [172]. In our studies, IL-2 receptor-decorated VLP not only bound but also neutralized soluble IL-2 [136]. IL-2 receptor-decorated VLP thereby displayed cytokine deprivation capabilities commonly associated with regulatory cells [136].…”

“…The prominent coreceptors CD4 and CD8 as well as a number of Src-related tyrosine kinases such as Lck or Fyn become fatty acid acylated and selectively sequestered into rafts to promote cellular signal transduction (table 1) [94,95,96,127,128,129,130,131,132,133]. Hence, in our work the genetic introduction of GPI anchor acceptor sequences at the C-terminal ends of the ectodomains of previously non-GPI-anchored proteins can efficiently target the immunomodulatory molecules of choice into the lipid rafts of the plasma membrane [134,135,136,137,138]. …”

“…GPI anchor attachment, leading to their concentration in lipid rafts of producer cells and the subsequent pseudotyping of emerging VLP. The specific composition, functional capabilities, and possible applications of such pseudotyped VLP, called ‘immunosomes’, ‘anergosomes’, and ‘fluorosomes’ (FS) [134,135,136,137], are shown in figure 1. …”

“…Notably, VLP can also be efficiently decorated with functionally active multimeric cytokine receptors such as the IL-2 receptor which consists of an α-, a β-, and a γ-chain (CD25, CD122, CD132) [136]. These findings seem to be of special interest since CD25+ regulatory CD4+ T cell (nTreg) subsets have been shown to exert their regulatory function on activated effector CD4+ T cells by – among other mechanisms – binding and neutralizing secreted IL-2 and other common γ-chain-(CD132)-associated cytokines [172].…”