Transcript Profiling of T Lymphocytes and Dendritic Cells in a Co–culture System Using Anti‐CD3 and Allergen Activation

Abstract

The interaction of antigen‐specific T lymphocytes with hapten‐bearing dendritic cells (DC) and the subsequent activation and clonal expansion of specific T lymphocyte populations are critical steps in the induction of skin sensitization. Therefore, we have sought to characterize changes in gene expression in T lymphocytes stimulated by incubation with allergen‐treated DC compared with anti‐CD3‐treated T cell‐DC cocultures as a method to identify potential markers of skin sensitization. Human T cells and autologous, mature peripheral blood‐derived DC were co–cultured in the presence or absence of anti‐CD3 monoclonal antibody (mAb) for 6 hours at a 10:1 responder:stimulator ratio. In a separate experiment, autologous DC and T cells from a donor sensitized to the potent contact allergen dinitrochlorobenzene (DNCB) were isolated. T cells were cultured for 6 hours at a responder to stimulator ratio of 20:1 with mature DC that had been treated with either 1 mM 2,4‐dinitrobenzenesulfonic acid (DNBS; the water soluble analog of DNCB), or media alone for 15 minutes. Total RNA was prepared and changes in gene expression were analyzed using Affymetrix U95Av2 GeneChips®. Comparative analysis of Affymetrix mean signal values from triplicate control cultures with those from anti‐CD3‐treated samples revealed highly significant (p ≤ 0.001) changes in the expression of 344 transcripts of the total of approximately 12,000 represented on the chip. However, mean signal values for T cells cocultured with allergen‐treated DC compared to vehicle‐treated DC‐T cell co–cultures identified only 17 significant gene changes (p ≤ 0.001), 11 of which were also identified as having changed significantly in response to stimulation with anti‐CD3. In parallel assays, antigen‐specific T cell proliferative responses were assessed as a function of tritiated thymidine incorporation. Increased T cell proliferative responses were observed in the cultures that contained both DNBS‐treated DC and T cells as well as the anti‐CD3 treated cultures compared with their respective controls. These data suggest that this approach can be used to identify genes that might serve as indicators of contact allergy and may be used in an in vitro predictive assay for skin sensitization.