Ozenci V; Kouwenhoven M; Huang Y; Xiao B; Kivisakk P; Fredrikson S; Link H, Multiple Sclerosis: Levels of Interleukin-10-Secreting Blood Mononuclear Cells are Low in Untreated Patients but Augmented During Interferon-b-1b Treatment. may be silenced by multivalent soluble antigen arrays (SAgAs), which are polymer conjugates displaying multiple copies of autoantigen with or without a secondary peptide that blocks intracellular cell-adhesion molecule-1 (ICAM-1). Here, key therapeutic molecular properties of SAgAs were identified and linked to the immunological mechanism through comprehensive cellular and analyses. We determined non-hydrolyzable cSAgAs displaying multivalent click-conjugated antigen more potently suppressed experimental autoimmune encephalomyelitis (EAE) compared to hydrolyzable SAgAs capable of releasing conjugated antigen. cSAgAs restored a healthy phenotype in disease-specific antigen presenting cells (APCs) by inducing an anergic response in B cells and a subset of B cells called autoimmune-associated B cells (ABCs) that act as potent APCs in autoimmune disease. Accompanied by a cytokine response skewed towards a Th2/regulatory phenotype, this generated an environment of autoantigenic tolerance. By identifying key therapeutic molecular properties and an immunological mechanism that drives SAgA efficacy, this work guides the design of antigen-specific immunotherapies capable of inducing anergy. in a murine model of MS (experimental autoimmune encephalomyelitis (EAE))24C28 and exhibited antigen-specific binding with B Rabbit Polyclonal to CACNG7 cells, targeted the B cell receptor (BCR), and dampened BCR-mediated signaling efficacy compared to hydrolyzable SAgAPLP:LABL.30 We concluded that non-hydrolyzable conjugation increased the avidity of cSAgAPLP:LABL to drive efficacy through dampened BCR-mediated signaling via a mechanism of sustained action (BCR binding and clustering) on the cell surface. Here, we identified SAgA immunological mechanisms using the EAE mouse model to define arising immune tolerance pathways to the PLP antigen used to induce the disease. Splenocyte immune responses were evaluated following and treatment with click-conjugated (non-hydrolyzable) and hydrolyzable soluble antigen arrays, mixtures of the components, and controls. Targeted immune cell subtypes were identified through flow cytometry binding assays and microfluidic imaging of live cells. Reduced costimulatory signaling was evaluated as a marker of anergy and lasting antigenic tolerance in EAE splenocytes following and treatment. Cytokines, PLP-specific Otenabant autoantibody production, and splenic immune cell composition were also evaluated following treatment to determine the shape of the effector response (Th1 vs. Th2, Treg vs. Th17). Through these comprehensive cellular and analyses, we determined that multivalent polymer arrays displaying click-conjugated PLP induced an anergic response in B cells and a subset of B cells that act as potent APCs in autoimmune disease called autoimmune-associated B cells (ABCs), accompanied by a cytokine response skewed towards a Th2/regulatory phenotype. By identifying cornerstone molecular properties and an immunological mechanism that drives SAgA efficacy, this work guides our understanding of the antigen-specific immune response and informs the future design of ASIT. 2.?MATERIALS AND METHODS 2.1. Materials Hyaluronic acid (HA) sodium salt (MW 16 kDa) was purchased from Lifecore Biomedical (Chaska, MN). 11-azido-3,6,9-trioxaundecan-1-amine (NH2-PEG3-N3), N-hydroxysuccinimide, strain H37RA were purchased from Otenabant Difco (Sparks, MD). Pertussis toxin was purchased from List Biological Laboratories (Campbell, CA). R-phycoerythrin (PE)/Cy7-conjugated anti-mouse CD3, AlexaFluor647-conjugated anti-mouse CD19, Pacific Blue-conjugated anti-mouse CD11c, PerCP-conjugated anti-mouse B220, PE-conjugated anti-mouse CD86, FITC-conjugated anti-mouse CD80, and respective isotype control antibodies were purchased from BioLegend (San Diego, CA). All other chemicals and reagents were analytical grade and used as received. 2.2. Synthesis of Click Soluble Antigen arrays (cSAgAs) Penn Green-Alk, HA-N3, and click soluble antigen arrays (cSAgAs) were prepared as previously reported.30 Briefly, cSAgAs were constructed using a two-step procedure starting from sodium hyaluronate. 3-(ethyliminomethyleneamino)-N,N-dimethylpropan-1-amine (EDC) and N-hydroxysuccinimide (NHS) neat were added to a solution of sodium hyaluronate in MES buffer. After Otenabant 5 min of stirring, H2N-PEG3-N3 was added and the solution was stirred at room temperature for 24 hrs before being dialyzed and lyophilized to isolate HA-N3. The appropriate ligands (hpPLP139C151, hpLABL, Penn Green-Alk) were added to a solution of HA-N3 in deionized water, followed by a premixed solution of tris(3-hydroxypropyl-triazolylmethyl)amine (THPTA) and copper (II) sulfate (CuSO4-5H2O) in deionized water. After stirring for 1C2 min, sodium ascorbate (NaAsc) was added and the reaction was allowed to proceed at elevated temperature until the desired conjugation levels were achieved. Following completion of the reaction, the reaction solution was dialyzed and lyophilized. cSAgAs were.