title: Cloning and large-scale expansion of epitope-specific equine cytotoxic T lymphocytes using an anti-equine CD3 monoclonal antibody and human recombinant IL-2.
abstract: Cytotoxic T lymphocytes are involved in controlling intracellular pathogens in many species, including horses. Particularly, CTL are critical for the control of equine infectious anemia virus (EIAV), a lentivirus that infects horses world-wide. In humans and animal models, CTL clones are valuable for evaluating the fine specificity of epitope recognition, and for adoptive immunotherapy against infectious and neoplastic diseases. Cloned CTL would be equally useful for similar studies in the horse. Here we present the first analysis of a method to generate equine CTL clones. Peripheral blood mononuclear cells were obtained from an EIAV-infected horse and stimulated with the EIAV Rev-QW11 peptide. Sorted CD8+ T cells were cloned by limiting dilution, and expanded without further antigen addition using irradiated PBMC, anti-equine CD3, and human recombinant IL-2. Clones could be frozen and thawed without detrimental effects, and could be subsequently expanded to numbers exceeding 2 x 10(9)cells. Flow cytometry of expanded clones confirmed the CD3+/CD8+ phenotype, and chromium release assays confirmed CTL activity. Finally, sequencing TCR beta chain genes confirmed clonality. Our results provide a reliable means to generate large numbers of epitope-specific equine CTL clones that are suitable for use in downstream applications, including functional assays and adoptive transfer studies.
authors.affiliation: Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164-7040, USA. rhm@vetmed.wsu.edu
authors.forename: Robert H
authors.initials: RH
authors.lastname: Mealey
authors.forename: Matt H
authors.initials: MH
authors.lastname: Littke
authors.forename: Steven R
authors.initials: SR
authors.lastname: Leib
authors.forename: William C
authors.initials: WC
authors.lastname: Davis
authors.forename: Travis C
authors.initials: TC
authors.lastname: McGuire
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D017252:CD3 Complex / Q000276:immunology*; D002999:Clone Cells / Q000166:cytology* / Q000187:drug effects; D018984:Epitopes, T-Lymphocyte / Q000276:immunology*; D006736:Horses / Q000276:immunology*; D006801:Humans; D007376:Interleukin-2 / Q000276:immunology* / Q000494:pharmacology; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins; D013602:T-Lymphocytes, Cytotoxic / Q000166:cytology* / Q000187:drug effects / Q000276:immunology*; D013997:Time Factors
chemical_list: D000911:Antibodies, Monoclonal; D017252:CD3 Complex; D018984:Epitopes, T-Lymphocyte; D007376:Interleukin-2; D016693:Receptors, Antigen, T-Cell, alpha-beta; D011994:Recombinant Proteins