Human T lymphocyte primingin vitroby haptenated autologous dendritic cells

Abstract

Dendritic cells (DC), generated from adherent peripheral blood mononuclear cells (PBMC) by culturing with granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4, were used to study in vitro sensitization of naive, hapten-specific T cells and to analyse cross-reactivities to related compounds. DC were hapten-derivatized with nickel sulphate (Ni) or 2-hydroxyethyl-methacrylate (HEMA), followed by tumour necrosis factor-alpha (TNF-alpha)-induced maturation, before autologous T cells and a cytokine cocktail of IL-1beta, IL-2 and IL-7 were added. After T cell priming for 7 days, wells were split and challenged for another 7 days with Ni or HEMA, and potentially cross-reactive haptens. Hapten-specificity of in vitro priming was demonstrated by proliferative responses to the haptens used for priming but not to the unrelated haptens. Highest priming efficiencies were obtained when both IL-4 and IL-12 were added to the cytokine supplement. Marked interferon-gamma (IFN-gamma) release (up to 4 ng/ml) was found when IL-12 was included in the cultures, whereas IL-5 release (up to 500 pg/ml) was observed after addition of IL-4 alone, or in combination with IL-12. Nickel-primed T cells showed frequent cross-reactivities with other metals closely positioned in the periodic table, i.e. palladium and copper, whereas HEMA-primed T cells showed distinct cross-reactivities with selected methacrylate congeners. Similar cross-reactivities are known to occur in allergic patients. Thus, in vitro T cell priming provides a promising tool for studying factors regulating cytokine synthesis, and cross-reactivity patterns of hapten-specific T cells.

Present and future ofin vitroimmunotoxicology in drug development

“…In vitro T-cell activation Dai and Streilein, 1998;Rustemeyer et al, 1999;Gorbachev and Fairchild, 2001 lysine gave a prediction accuracy of 89%. The results of these investigations revealed that measurement of peptide reactivity has considerable potential utility as a screening approach for skin sensitization testing, and thereby for reducing reliance on animal-based test methods (Gerberick et al, 2007b(Gerberick et al, , 2008.…”

“…An effective system could be to expand the absolute number of antigen-specific T-lymphocytes from the naïve T-lymphocyte pool of peripheral blood samples. The system has successfully applied to determine T-lymphocyte frequencies for several allergens in human blood samples and for activation of naive hapten-specific T-lymphocytes to study the earliest T-lymphocyte and APC events in the induction of human contact hypersensitivity (Dai and Streilein, 1998;Rustemeyer et al, 1999).…”

Copper and its inorganic compounds [MAK Value Documentation, 2006]

“…After in vitro induction with nickel sulfate, it was possible to stimulate quite a number of T cell clones from persons not sensitized to nickel with copper chloride. T cell clones not having received pre-treatment were not investigated (Rustemeyer et al 1999).…”

T-cell recognition of chemicals, protein allergens and drugs: towards the development of in vitro assays

“…Efforts were made to study primary human T-cell responses to chemicals. Different approaches were taken to induce T-cell responses to chemicals with respect to the populations of T cells and APC used, the means of application of the test chemicals and the cell culture protocols and readouts [42][43][44][45][46][47][48][49][50][51]. Table 2 summarizes the studies that have used protocols for priming of naive T cells with chemicals applied as shown in Fig.…”

“…T cells in the presence of various cytokine cocktails [47,49]. After 10-14 days T cells were divided into three aliquots and restimulated with medium alone, with the allergen used for priming or with a noncrossreacting or crossreacting contact allergen.…”