Insect cells as HLA-restricted @entity1358 for the XXXX elispot assay.

@entity1 @entity137 @entity320 @entity5 @entity1358 @entity379 @entity405 @entity238
Measurement of specific cellular immune responses in @entity1 undergoing immunotherapy is difficult. Established approaches, including @entity137 (e.g., 51Cr release) and cytokine release assays, require in vitro culturing for several weeks or more of @entity1 ' peripheral blood mononuclear cells (PBMC) and the addition of exogenous cytokines. Therefore, the immunological response does not reflect in vivo conditions. To address these disadvantages, we have used an @entity379 ( @entity379 ) Elispot assay for detecting peptide-specific @entity405 (+) lymphocytes in PBMC. A limitation of this assay is the lack of a reproducible source of @entity1358 ( @entity1358 ). Currently available @entity1358 often lead to significant background levels. It has been shown that transfected insect cells can express empty MHC class I molecules on their surface. We have transfected @entity238 S2 cells and the Lepidopteran line Sf9 with the gene encoding @entity1 HLA-A2.1. We demonstrate that insect cells expressing a @entity1 HLA molecule effectively function as @entity1358 in the @entity379 Elispot assay. Initially the feasibility of the assay was assessed using @entity405 (+) T cells from HLA-A2.1(+) donors with known reactivity against an HLA-A2.1-binding epitope of the influenza matrix protein. Use of insect cells as @entity1358 abrogated background spots, increasing sensitivity. We further observed that a short-term prestimulation of PBMC with peptide-pulsed insect cells markedly enhanced the frequency of peptide-specific T cells that could be measured in the Elispot assay without increasing the background. This approach was then used to measure @entity405 (+) T cell reactivity to a peptide from tyrosinase, an antigen that is processed and presented by @entity320 cells. Insect cells expressing @entity1 HLA molecules provide a standard APC for monitoring @entity405 (+) T cell responses to @entity5 and viral peptides during immunotherapy.
