Relative percentages of cells were quantified for GL7+ CD19+ GC-like B cells following culture in hydrogel functionalized with either REDV (blue) or RGD (red) in the presence of anti-41 antibody or Cilengitide anti-v3 peptide) added at various time points in culture
Relative percentages of cells were quantified for GL7+ CD19+ GC-like B cells following culture in hydrogel functionalized with either REDV (blue) or RGD (red) in the presence of anti-41 antibody or Cilengitide anti-v3 peptide) added at various time points in culture. that represents a recombined antibody variable region derived from a 4-hydroxy-3-nitrophenylacetyl (NP) hapten binding antibody (B1C8), we demonstrate antigen specificity and selective enrichment of antigen-specific B cells with high affinity at both cell surface and secreted levels in integrin ligand-dependent manner. The antigen-specific platform technology offers use in scientific understanding of immunobiology, matrix immunology, and in biotechnology applications, ranging from the antigen testing, Cd300lg vaccine development, and generation of antibodies against diseases. Keywords: Contamination, Immunity, Hydrogel, Tissue engineering, Integrin, Antibody 1.?Introduction Upon T cell-dependent activation, na?ve B cells form germinal center (GCs) and undergo massive proliferation, somatic hyper-mutation, and affinity-based selection prior to differentiation into Terlipressin memory B cells [1C5]. Despite our deep understanding of immunity, it is difficult to study modulators of GC since natural GCs are heterogeneous and composed of B cells constantly recycling between dark and light zones of GCs. GC B cells with low expression of C-X-C Chemokine Receptor Type 4 (CXCR4) and high expression of CD83 surface marker are found in the less proliferative light zone of GCs, where the selection process occurs ahead of GC exit for terminal differentiation [4]. In contrast, GC B cells undergoing proliferation in the dark zone exhibit higher expression of CXCR4 surface marker and low expression of CD83 surface markers [4]. GC B cells [5,14]. Therefore, there is an unmet need for an technology capable of recapitulating selective complexity of GC reaction and induce antigen-specific humoral immunity. We recently reported a gelatin-based 3D immune tissue that recapitulated selective cell-cell signaling aspects of the GC process and presented an RGD-rich niche [14C16]. The tissue-derived GC B cells showed amazing similarity to GC B cells from immunized mice in terms of the GC phenotype, transcriptome, induction of the GC grasp regulators B Cell CLL/Lymphoma 6 (BCL6) and the Enhancer of zeste homolog 2 (EZH2) histone methyltransferase, as well as activation-induced cytidine deaminase and somatic hypermutation [14]. Furthermore, the RGD-rich tissue recapitulated the GC-specific conditional deletion of EZH2 [14]. Using this tissue model we exhibited that EZH2, which represses gene expression by catalyzing histone 3 lysine 27 trimethylation (H3K27me3), mediates GC formation through epigenetic silencing of the cell cycle checkpoint gene cyclin dependent kinase inhibitor 1A (CDKN1A) [14], enabling a positive transcriptional feedback loop. These studies, however, did not demonstrate antigen speci-ficity, as noted in a recent review by Gosselin et al. [17], where T-cell signal becomes critical for selective enrichment. Here, we report a maleimide (MAL)-functionalized polyethylene glycol (PEG)-based designer immune tissues for regulating GC B cell phenotype, including CXCR4 and CD83, B cell differentiation signaling, and selective enrichment of antigen-specific GC B cells using a first apoptosis signal (FAS)-based approach. 2.?Materials and methods 2.1. Biomaterials and peptides Four-arm PEG-MAL with 20 kDa molecular weight was obtained from Laysan Bio (Arab, AL) with >90% purity. Integrin avb3-binding RGD peptide (GRGDSPC, >90% purity), scrambled peptide (GRDGSPC, >90% purity) integrin 41-binding REDV peptide (GREDVGC, >90% purity), and matrix metalloproteinases (MMP)-9 degradable VPM peptide (GCRDVPMSMRGGDRCG, >90% purity) were obtained from AAPPTec (Louisville, KY). Integrin v3 inhibitor Cilengitide (a cyclic RGD) was obtained from Selleck Chemicals. 2.2. Immune cells For experiments involving wildtype (WT) B cells, spleens were harvested from female C57BL/6 mice (Stock #: 000664), aged 10C18 weeks from the Jackson Laboratory (Bar Harbor, ME). Terlipressin For experiments utilizing antigen-specific B cells, spleens were obtained from male B1C8hi mice (Stock #: 007594), aged 10C12 weeks from the Jackson Laboratory. For experiments involving conditional knockout(KO) mice, spleens were obtained from C1-cre+ GC reaction. Open in a separate windows Fig. 1. Designer immune tissues control GC-like B cell phenotype.A) Schematic Terlipressin showing PEG-MAL hydrogels functionalized with REDV or RGD peptides and embedded with na?ve B cells and 40LB stromal cells.