title: Application of IL-5 ELISPOT assays to quantification of antigen-specific T helper responses.
abstract: To be able to determine the frequencies of helper-type T lymphocyte (Th) precursors specific for viral or tumor-associated antigens, an ELISPOT assay for IL-5 production was developed. The assay reproducibility was first determined using fresh or cryopreserved peripheral blood mononuclear cells (PBMC) obtained from six normal donors and tested for IL-5 production after 48 h stimulation with phytohemagglutinin (PHA). Both inter-assay (within-subject CV=1.6%) and intra-assay (within-subject CV=0.8%) variabilities were found to be acceptable, and the frequencies of IL-5 secreting cells were comparable in cryopreserved and fresh PBMC of the same donors. The presence and frequency of Th precursor cells to viral capsid L1 protein (viral-like particles, VLP-L1) of human papillomavirus type 16 (HPV-16) in PBMC obtained from seven non-immunized donors and two volunteers immunized with VLP-L1 were then evaluated. Using autologous dendritic cells as antigen-presenting cells (APC) for VLP-L1 in direct 48-h ELISPOT assays, the mean frequency of IL-5 secreting CD4+ T cells was found to be <1/10(5) (negative) in normal donors but was 1/2, 436 and 1/3678 in the two volunteers immunized with VLP-L1. The assay is applicable to monitoring of the frequency of antigen-specific T cells in the peripheral circulation of individuals immunized with HPV-derived antigens. To test the assay utility in the assessment of Th tumor-reactive lymphocytes, we also used it to determine the frequency of the wild-type sequence (wt) p53(22-36) peptide-specific, HLA-DR4-restricted T cells in the bulk CD4+ T cell line. This frequency was 1/33. The ELISPOT assay for IL-5 production can be reliably used to measure Th-type responses in a variety of experimental settings.
authors.affiliation: University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213-2582, USA.
authors.forename: Jaafar
authors.initials: J
authors.lastname: Bennouna
authors.forename: Allan
authors.initials: A
authors.lastname: Hildesheim
authors.forename: Kazuaki
authors.initials: K
authors.lastname: Chikamatsu
authors.forename: William
authors.initials: W
authors.lastname: Gooding
authors.forename: Walter J
authors.initials: WJ
authors.lastname: Storkus
authors.forename: Theresa L
authors.initials: TL
authors.lastname: Whiteside
mesh_terms: D000956:Antigens, Viral; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D036022:Capsid Proteins; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D015804:HLA-DR4 Antigen; D006801:Humans; D007114:Immunization; D066298:In Vitro Techniques; D015848:Interleukin-5 / Q000032:analysis* / Q000096:biosynthesis; D009856:Oncogene Proteins, Viral / Q000276:immunology; D027383:Papillomaviridae / Q000276:immunology; D015203:Reproducibility of Results; D006377:T-Lymphocytes, Helper-Inducer / Q000276:immunology*; D016159:Tumor Suppressor Protein p53 / Q000276:immunology
chemical_list: D000956:Antigens, Viral; D036022:Capsid Proteins; D015804:HLA-DR4 Antigen; D015848:Interleukin-5; D009856:Oncogene Proteins, Viral; D016159:Tumor Suppressor Protein p53; C063317:L1 protein, Human papillomavirus type 16