title: A novel ELISPOT assay to enhance detection of antigen-specific T cells employing antigen-presenting cells expressing vector-driven human B7-1.
abstract: The ELISPOT assay is used to detect T-cell responses in patients enrolled in vaccine clinical trials; it is a relatively sensitive assay that can be performed without in vitro stimulation (IVS) of PBMC. However, this assay may be limited in some cases because of a weak signal 1 (as is the case for tumor-associated antigens [TAA]), or by a limited number of PBMC available from patients. The objective of this study is to enhance the sensitivity of the ELISPOT by increasing the level of signal 2, in this case, B7-1 expression on antigen-presenting cells (APC), allowing for a more sensitive detection of antigen-specific T-cell precursors. C1RA2 cells were used as APC and were uninfected, or infected with either recombinant avipox (fowlpox)-B7-1 (rF-B7-1) or control fowlpox wild-type (FP-WT) vector. The expressions of B7-1, MHC Class II, as well as HLA-A2 on the infected cells were analyzed by flow cytometry. A nearly threefold increase in B7-1 mean fluorescence intensity (MFI) occurred in the rF-B7-1-infected C1RA2 cells with no changes in MHC Class II or HLA-A2 expression. Various PBMC/APC ratios were used to further analyze the use of rF-B7-1-infected C1RA2 cells as APC in the ELISPOT assay. Fewer APC were required to activate PBMC when C1RA2 infected with rF-B7-1 were used as APC. Furthermore, using a PBMC/APC ratio of 1:1, similar detection was achieved using fewer PBMC. In addition, we demonstrated that the reactivity can be blocked by adding anti-B7-1 antibody. We performed the assay using APC on five normal healthy donors. All five donors showed substantial increases in PF to the Flu matrix peptide (Flu peptide) using the rF-B7-1-infected C1RA2 cells. Finally, we evaluated five cancer patients who received a carcinoembryonic antigen (CEA) vaccine-based therapy. Increases in CEA peptide precursors were noted in all five patients using the B7-1-infected APC. In conclusion, we have demonstrated the ability to enhance the sensitivity of the ELISPOT assay through infection of C1RA2 with rF-B7-1.
authors.affiliation: National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Philip M
authors.initials: PM
authors.lastname: Arlen
authors.forename: James L
authors.initials: JL
authors.lastname: Gulley
authors.forename: Claudia
authors.initials: C
authors.lastname: Palena
authors.forename: John
authors.initials: J
authors.lastname: Marshall
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Schlom
authors.forename: Kwong Yok
authors.initials: KY
authors.lastname: Tsang
mesh_terms: D000938:Antigen-Presenting Cells / Q000378:metabolism*; D018122:B7-1 Antigen / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D018014:Gene Transfer Techniques; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism; D013601:T-Lymphocytes / Q000378:metabolism*
chemical_list: D018122:B7-1 Antigen; D007371:Interferon-gamma