Analysis of the Cellular Immune Responses to Vaccines

• Published in: Methods in molecular biology (Clifton, N.J.) Vol. 1349; p. 247
• Main Authors: Svitek, Nicholas, Taracha, Evans L N, Saya, Rosemary, Awino, Elias, Nene, Vishvanath, Steinaa, Lucilla
• Format: Journal Article
• Language: English
• Published: United States 2016
• Subjects: Amino Acid Sequence - genetics; Animals; Cattle; Enzyme-Linked Immunospot Assay - methods; Epitopes, T-Lymphocyte - immunology; Flow Cytometry - methods; Immunity, Cellular - genetics; Immunity, Cellular - immunology; Livestock - virology; Peptides - immunology; T-Lymphocytes, Cytotoxic - immunology; Vaccines - immunology; Cytotoxicity assay; Flow cytometry; Peptide-MHC class I tetramers; CTL epitope; NetMHCpan; ELISpot
• ISSN: 1940-6029
• DOI: 10.1007/978-1-4939-3008-1_16

Flow cytometry, enzyme-linked immunospot (ELISpot) and cellular cytotoxicity assays are powerful tools for studying the cellular immune response towards intracellular pathogens and vaccines in livestock species. Lymphocytes from immunized animals can be purified using Ficoll-Paque density gradient centrifugation and evaluated for their antigen specificity or reactivity towards a vaccine. Here, we describe staining of bovine lymphocytes with peptide (p)-MHC class I tetramers and antibodies specific towards cellular activation markers for evaluation by multiparametric flow cytometry, as well as interferon (IFN)-γ ELISpot and cytotoxicity using chromium ((51)Cr) release assays. A small component on the use of immunoinformatics for fine-tuning the identification of a minimal CTL epitope is included.