Human T lymphocyte priming in vitro by 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.

Fig. 1

In vitro priming of T cells is hapten-specific. After culturing 1 × 10⁵ T cells with nickel sulphate (Ni)- or 2-hydroxyethyl-methacrylate (HEMA)-loaded dendritic cells (DC) for 7 days, wells were split and challenged with Ni, HEMA or medium only. Proliferation was measured after 7 days. (a) For donor 1, ³H-thymidine uptake data (ct/min) are given; wells showing a positive stimulation index (SI) (ratio of ³H-thymidine uptake by hapten-challenged T cells to the uptake in medium control cultures > 2.0 and Δ ct/min > 500) are indicated by the actual SI values. (b) For four donors, donor 1 (▪) and three other donors (▴,•,▾), SI are presented; each symbol represents one well; closed symbols indicate wells showing a positive SI.

Fig. 2

T cell priming in presence of IL-4 and/or IL-12 determines the cytokine production profile. Nickel sulphate-loaded dendritic cells (DC) were cultured with 1 × 10⁵ T cells, in presence or absence of IL-4 and/or IL-12. After 7 days, wells were split and challenged with nickel sulphate and medium. The production of IL-5 and IFN-γ in the culture supernatants and cellular proliferation were measured after 7 days. Figures show results of three donors, each bar represents one well.

Fig. 3

Primary in vitro induction of hapten-specific T cell responses is restricted to CD45RA⁺ T cells. Isolated CD45RA⁺ and CD45RO⁺ T cells (1 × 10⁵) of two non-sensitized donors were cultured with nickel sulphate-loaded dendritic cells (DC) in the presence of IL-1β, IL-2, IL-4, IL-7 and IL-12 for 7 days. Subsequently, wells were split and cultured further with nickel sulphate and medium. Proliferation was measured after 7 days and stimulation indices (SI) are expressed as the ratio of ³H-thymidine uptake by hapten-challenged T cells to the uptake in medium control cultures. Figure shows results of two donors (○,▪); each symbol represents one well, positive wells (SI > 2.0 and Δ > 500 ct/min) are indicated by closed symbols.

Fig. 4

The precursor frequency of nickel-specific T cells in naive donors is below one specific cell per 2 × 10⁶ T cells. Thirty thousand to 1 × 10⁶ T cells of three non-sensitized donors were cultured with nickel sulphate-loaded dendritic cells (DC) (DC:T cell ratio 1:10) in the presence of IL-1β, IL-2, IL-4, IL-7 and IL-12 for 7 days. Subsequently, wells were split and cultured further with nickel sulphate and medium. Proliferation was measured after 7 days and stimulation indices (SI) are expressed as the ratio of ³H-thymidine uptake by hapten-challenged T cells to the uptake in medium control cultures. For three donors (•,▾,▪) the relative number of negative wells (SI < 2.0 and Δ < 500 ct/min) and the linear regression analysis is presented (for •– · — · –; for ▾—-; for ▪ - - - - -).

Fig. 5

(a) Nickel sulphate-primed T cells can show cross-reactions to copper and palladium and (b) 2-hydroxyethyl-methacrylate (HEMA)-primed T cells to methacrylate congeners. Isolated T cells (1 × 10⁵) were cultured with nickel sulphate- or HEMA-loaded dendritic cells (DC) in the presence of IL-1β, IL-2, IL-4, IL-7 and IL-12 for 7 days. Subsequently, wells were split and cultured further, upon priming with nickel sulphate (a), with medium, nickel sulphate (•) and palladium (Pd, ▾), copper (Cu, ▴) or HEMA (▪), and, upon priming with HEMA (b), with medium, HEMA (•) and methyl methacrylate (MMA, ▾), 2-hydroxypropyl methacrylate (HPMA, ▴), ethyleneglycol dimethacrylate (EGDMA, ▪) or nickel sulphate (○). Proliferation was measured after 7 days and stimulation indices (SI) are expressed as the ratio of ³H-thymidine uptake by hapten-challenged T cells to the uptake in medium control cultures. Summarized data of five donors; each symbol represents one well, positive wells (SI > 2.0 and Δ > 500 ct/min) are indicated by closed symbols.