Insect cells as HLA-restricted antigen-presenting cells for the IFN-gamma Elispot assay

Measurement of specific cellular immune responses in patients undergoing im munotherapy is difficult. Established approaches, including cytotoxicity (e .g., Cr-51 release) and cytokine release assays, require in vitro culturing for several weeks or more of patients' peripheral blood mononuclear cells (PBMC) and the addition of exogenous cytokines. Therefore, the immunologica l response does not reflect in vivo conditions. To address these disadvanta ges, we have used an interferon-gamma (IFN-gamma) Elispot assay for detecti ng peptide-specific CD8(+) lymphocytes in PBMC. A limitation of this assay is the lack of a reproducible source of antigen-presenting cells (APCs). Cu rrently available APCs often lead to significant background levels. It has been shown that transfected insect cells can express empty MHC class I mole cules on their surface. We have transfected Drosophila melanogaster S2 cell s and the Lepidopteran line Sf9 with the gene encoding human HLA-A2.1. We d emonstrate that insect cells expressing a human HLA molecule effectively fu nction as APCs in the IFN-gamma Elispot assay. Initially the feasibility of the assay was assessed using CD8(+) T cells from HLA-A2.1(+) donors with k nown reactivity against an HLA-A2.1-binding epitope of the influenza matrix protein. Use of insect cells as APCs abrogated background spots, increasin g sensitivity. We further observed that a short-term prestimulation of PBMC with peptide-pulsed insect cells markedly enhanced the frequency of peptid e-specific T cells that could be measured in the Elispot assay without incr easing the background. This approach was then used to measure CD8(+) T cell reactivity to a peptide from tyrosinase, an antigen that is processed and presented by melanoma cells. Insect cells expressing human HLA molecules pr ovide a standard APC for monitoring CD8(+) T cell responses to tumor and vi ral peptides during immunotherapy. (C) 2000 Elsevier Science B.V. All right s reserved.