Notch-ligand expression by NALT dendritic cells regulates mucosal Th1- and Th2-type responses

Abstract

Our previous studies showed that an adenovirus (Ad) serotype 5 vector expressing Flt3 ligand (Ad-FL) as nasal adjuvant activates CD11c(+) dendritic cells (DCs) for the enhancement of antigen (Ag)-specific IgA antibody (Ab) responses. In this study, we examined the molecular mechanism for activation of CD11c(+) DCs and their roles in induction of Ag-specific Th1- and Th2-cell responses. Ad-FL activated CD11c(+) DCs expressed increased levels of the Notch ligand (L)-expression and specific mRNA. When CD11c(+) DCs from various mucosal and systemic lymphoid tissues of mice given nasal OVA plus Ad-FL were cultured with CD4(+) T cells isolated from non-immunized OVA TCR-transgenic (OT II) mice, significantly increased levels of T cell proliferative responses were noted. Furthermore, Ad-FL activated DCs induced IFN-γ, IL-2 and IL-4 producing CD4(+) T cells. Of importance, these APC functions by Ad-FL activated DCs were down-regulated by blocking Notch-Notch-L pathway. These results show that Ad-FL induces CD11c(+) DCs to the express Notch-ligands and these activated DCs regulate the induction of Ag-specific Th1- and Th2-type cytokine responses.

Fig. 1

Analysis of DC function for induction of CD4 T cell proliferation. Mice were nasally immunized weekly for three consecutive weeks with OVA plus Ad-FL as mucosal adjuvant or Ad-Luc as a vector control. One week after the last immunization, CD11c⁺ DCs were purified from NALT, CLNs, NPs, SMGs and spleens, and cultured with naïve splenic CD4⁺ T cells isolated from non-immunized OVA TCR-transgenic (OT II) mice with or without 1 mg/ml of OVA for 5 days. An aliquot of 0.5 μCi of tritiated [³H]TdR was added during the final 18 h of incubation. The results are presented as the cpm of wells with OVA following subtraction of the cpm of wells without OVA. * p < 0.05 when compared with mice immunized with Ad-Luc plus OVA.

Fig. 2

Expression of Notch ligand-specific mRNA. Mice were nasally immunized weekly for three consecutive weeks with Ad-FL plus OVA. One week after the last immunization, CD11c⁺ DCs were purified from NALT (A), CLNs (B), NPs (C), SMGs (D) and spleens (E). Total RNA was subjected to quantitative RT-PCR analysis. The values shown are the mean ± SEM taken from 25 mice in each experimental group. * p < 0.05 when compared with mice immunized with Ad-Luc plus OVA.

Fig. 3

Analysis of Th1- and Th2-cytokine production by OVA-specific CD4⁺ T cells. Mice were nasally immunized as described in Fig. 1 legend. One week after the last immunization, CD11c⁺ DCs were purified from NALT, CLNs, NPs, SMGs and spleens, and cultured with naïve splenic CD4⁺ T cells isolated from non-immunized OVA TCR-transgenic (OT II) mice with or without 1 mg/ml of OVA for 2 or 5 days. Culture supernatants were harvested after 5 days of incubation and analyzed for the respective cytokine by ELISA. Total RNA was extracted after 2 days of incubation and subjected to quantitative RT-PCR analysis. The values shown are the mean ± SEM taken from 25 mice in each experimental group. * p < 0.05 when compared with mice given nasal Ad-Luc plus OVA.

Fig. 4

A blockade of the Notch-Notch-L pathway down-regulates the numbers of IFN-γ- and IL-4-producing T cells. Mice were nasally immunized as described in Fig. 1 legend. Purified CD11c⁺ DCs and splenic CD4⁺ T cells from OT II mice were co-cultured with 1 mg/ml of OVA in the presence or absence of DAPT for 5 days. Cells were stained with FITC conjugated anti-CD4 mAb followed by additional intracellular staining with PE-tagged anti-IFN-γ or -IL-4 mAbs. Samples were subjected to flow cytometric analysis by FACSCalibur®. The values shown are the mean ± SEM of 10 mice in each experimental group. *p < 0.05 when compared with the cultures in the absence of DAPT.