Computational protocol: Application of IgG-Derived Natural Treg Epitopes (IgG Tregitopes) to Antigen-Specific Tolerance Induction in a Murine Model of Type 1 Diabetes

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Protocol publication

[…] Draining inguinal and popliteal nodes were harvested and the samples were evaluated by FACS for T cell proliferation and Treg expansion. The KJ1-26 mAb that reacts with the clonotypic TCR expressed by DO11.10 transgenic mice was used to identify T cells expressing the transgenic OVA-specific TCR. To detect FoxP3/GFP+ Tregs, a single-cell suspension of draining lymph nodes was incubated with 2.4G2 mAb (anti-CD16/32, ATCC) for 15 minutes to block FcR, then stained with CD4-PE and anti-clonotypic KJ1-26-APC for 40 minutes at 4°C. Cells were acquired on a FACSCalibur flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA) and analyzed using FlowJo software (Treestar, Ashland, OR, USA). The CD4+KJ1-26+ live cell gate population was established, and these gated cells were analyzed for the presence of GFP fluorescence indicative of FoxP3 expression. Antigen-specific T cell proliferation was evaluated by [3H] thymidine incorporation []. Draining lymph nodes were harvested and a single-cell suspension was prepared at 2 × 106 cells/mL. Cells were added to 96-well plates at 100 μL per well in the presence of indicated concentration of OVA 323–339 (New England Peptide, Gardner, MA, USA). Forty-eight hours later, the cells were pulsed with 1 μCi/well of [3H] thymidine and incubated for another 16–20 hours. Cells were then harvested on glass fiber filters, and [3H] incorporation into the DNA was measured using a MicroBeta2 plate counter. [...] Of the 16 GAD65 peptides selected for this study, 14 could be synthesized and purified to ≥80% pure; GAD65 324-342 was not included in the studies reported here due to technical difficulties in peptide synthesis, and GAD65 108-131 was not included due to low purity. Therefore, we validated 14 epitopes in HLA-DR binding studies for use in ex vivo two-step ELISpot assays with human PBMC. Human Tregitopes and GAD65 epitopes were evaluated for binding to HLA in vitro. We used an HLA binding assay initially described by Steere et al. [] and adapted for higher throughput in our laboratory. In 96-well plates, a test peptide and a reference peptide competed for binding to purified class II HLA molecules (provided by William Kwok, Benaroya Institute, Seattle, WA, USA) for 24 hours at 37°C. The nonbiotinylated test peptides were evaluated over a range of concentrations, while the biotinylated reference peptide was held at a fixed concentration (0.1–1 μM depending on the HLA). The reference peptides and concentrations were as follows: biotin-Flu-HA 306–318 (PRYVKQNTLKLAT) at 0.1 μM for DRB1*0101 and DRB1*0401, and at 1 μM for DRB1*0701; and biotin-MBP 84–102 (NPVVHFFKNIVTPRTPPPS) at 0.5 μM for DRB1*1501. The peptides that bound to class II molecules were then captured on ELISA plates using pan anti-class II antibodies (L243, anti-HLA-DR alpha chain). The assays were developed by addition of streptavidin-europium and read on a time-resolved fluorescence plate reader. Standard curve analysis using four-parameter logistics was performed in SigmaPlot software, and an IC50 value was calculated for each peptide-allele pair in which 50% inhibition was encompassed by the range of test peptide dilutions (). Some peptides were available in limited supply and thus were only tested for binding to DRB1*0401. […]

Pipeline specifications

Software tools FlowJo, SigmaPlot
Applications Miscellaneous, Flow cytometry
Organisms Mus musculus, Homo sapiens
Diseases Autoimmune Diseases, Diabetes Mellitus, Diabetes Mellitus, Type 1, Hyperglycemia