Human dendritic cells activated by TSLP and CD40L induce proallergic cytotoxic T cells

Abstract

Human thymic stromal lymphopoietin (TSLP) is a novel epithelial cell-derived cytokine, which induces dendritic cell (DC)-mediated CD4+ T cell responses with a proallergic phenotype. Although the participation of CD8+ T cells in allergic inflammation is well documented, their functional properties as well as the pathways leading to their generation remain poorly understood. Here, we show that TSLP-activated CD11c+ DCs potently activate and expand naive CD8+ T cells, and induce their differentiation into interleukin (IL)-5 and IL-13-producing effectors exhibiting poor cytolytic activity. Additional CD40L triggering of TSLP-activated DCs induced CD8+ T cells with potent cytolytic activity, producing large amounts of interferon (IFN)-gamma, while retaining their capacity to produce IL-5 and IL-13. These data further support the role of TSLP as initial trigger of allergic T cell responses and suggest that CD40L-expressing cells may act in combination with TSLP to amplify and sustain pro-allergic responses and cause tissue damage by promoting the generation of IFN-gamma-producing cytotoxic effectors.

Figure 1.

CD11c⁺ DCs activated by TSLP alone or in combination with CD40L induce a strongly expansion of naive CD8⁺ T cells. (A) Naive CD8⁺ T cells were cultured for 6 d with CD11c⁺ DCs activated for 24 h with medium, CD40L, TSLP, or TSLP + CD40. TSLP-DCs induced stronger expansion of naive CD8⁺ T cells than CD40-L-DCs. The combination of TSLP and CD40L showed an additive effect on the capacity of activated DCs to induce CD8⁺ T cell expansion. Data represent five independent experiments; horizontal bars represent the median value. (B) Surface expression of CD80 and CD86 by medium-DCs, CD40L-DCs, TSLP-DCs, and TSLP+CD40L-DCs. TSLP-DCs showed significantly higher levels of CD80 than CD40L-DCs, TSLP+CD40L-DCs showed the highest levels of both CD80 and CD86. Filled histograms represent staining of costimulatory molecules CD80 and CD86; open histograms represent the isotype control. Numbers indicate the mean fluorescence intensity.

Figure 1.

CD11c⁺ DCs activated by TSLP alone or in combination with CD40L induce a strongly expansion of naive CD8⁺ T cells. (A) Naive CD8⁺ T cells were cultured for 6 d with CD11c⁺ DCs activated for 24 h with medium, CD40L, TSLP, or TSLP + CD40. TSLP-DCs induced stronger expansion of naive CD8⁺ T cells than CD40-L-DCs. The combination of TSLP and CD40L showed an additive effect on the capacity of activated DCs to induce CD8⁺ T cell expansion. Data represent five independent experiments; horizontal bars represent the median value. (B) Surface expression of CD80 and CD86 by medium-DCs, CD40L-DCs, TSLP-DCs, and TSLP+CD40L-DCs. TSLP-DCs showed significantly higher levels of CD80 than CD40L-DCs, TSLP+CD40L-DCs showed the highest levels of both CD80 and CD86. Filled histograms represent staining of costimulatory molecules CD80 and CD86; open histograms represent the isotype control. Numbers indicate the mean fluorescence intensity.

Figure 2.

TSLP-activated DCs induce IL-5 and IL-13–producing CD8⁺ T cells. Cytokine production by naive CD8⁺ T cells primed for 6 d with medium-DCs, CD40L-DCs, TSLP-DCs, or TSLP+CD40L-DCs. Harvested CD8⁺ T cells were restimulated for 24 h with anti-CD3 and anti-CD28 and IFN-γ, TNF-α, IL-4, IL-5, IL-10, and IL-13 were measured in the culture supernatant by ELISA. TSLP-DCs and TSLP+CD40L-DCs both induced IL-5 and IL-13 producing CD8⁺ T cells. However, in contrast to TSLP-DCs, TSLP+CD40L-DCs primed CD8⁺ T effector cells to produce high levels of IFN-γ. Data represent one of three independent experiments.

Figure 3.

CD40L-TSLP-DCs induced the generation of CD8⁺ cytotoxic effector T cells. (A) Naive CD8⁺ T cells were primed for 6 d with medium-DCs (triangles), CD40L-DCs (diamonds), TSLP-DCs (circles), or TSLP+CD40L-DCs (squares), collected and tested for their ability to lyse allogeneic HLA-A2–positive target cells. Specific lysis of autologous HLA-A2–negative target cells remained below 15%. Whereas both TSLP-DCs and CD40L-DCs induced CD8⁺ T cells with poor cytolytic activity, TSLP+CD40L-DCs induced CD8⁺ T cells with potent cytolytic activity. Data represent one of three independent experiments. (B) Two color staining for intracellular perforin and surface CD8 of T cells primed with medium-DCs, CD40-L-DCs, TSLP-DCs, or TSLP+CD40-L-DCs for 6 d.

Figure 3.

CD40L-TSLP-DCs induced the generation of CD8⁺ cytotoxic effector T cells. (A) Naive CD8⁺ T cells were primed for 6 d with medium-DCs (triangles), CD40L-DCs (diamonds), TSLP-DCs (circles), or TSLP+CD40L-DCs (squares), collected and tested for their ability to lyse allogeneic HLA-A2–positive target cells. Specific lysis of autologous HLA-A2–negative target cells remained below 15%. Whereas both TSLP-DCs and CD40L-DCs induced CD8⁺ T cells with poor cytolytic activity, TSLP+CD40L-DCs induced CD8⁺ T cells with potent cytolytic activity. Data represent one of three independent experiments. (B) Two color staining for intracellular perforin and surface CD8 of T cells primed with medium-DCs, CD40-L-DCs, TSLP-DCs, or TSLP+CD40-L-DCs for 6 d.