OX40 induces CCL20 expression in the context of antigen stimulation: an expanding role of co-stimulatory molecules in chemotaxis

Abstract

OX40 is an inducible co-stimulatory molecule expressed by activated T cells. It plays an important role in the activation and proliferation of T lymphocytes. Recently, some co-stimulatory molecules have been shown to direct leukocyte trafficking. Chemotaxis is essential for achieving an effective immune response. CCL20 is an important chemoattractant produced by activated T cells. In this study, using DO11.10 mice whose transgenic T cell receptor specifically recognizes ovalbumin, we demonstrate that ovalbumin induces OX40 expression in CD4+ lymphocytes. Further stimulation of OX40 by OX40 activating antibody up-regulates CCL20 production. Both NF-kappaB dependent and independent signaling pathways are implicated in the induction of CCL20 by OX40. Finally, we primed the DO11.10 splenocytes with or without OX40 activating antibody in the presence of ovalbumin. Intranasal administration of the cell lysates derived from the cells with OX40 stimulation results in more severe leukocyte infiltration in the lung of DO11.10 mice, which is substantially attenuated by CCL20 blocking antibody. Taken together, this study has shown that activation of OX40 induces CCL20 expression in the presence of antigen stimulation. Thus, our results broaden the role of OX40 in chemotaxis, and reveal a novel effect of co-stimulatory molecules in orchestrating both T cell up-regulation and migration.

Fig. 1

OVA induces OX40 expression primarily in CD4+ T cells in DO11.10 splenocytes. Splenocytes were isolated from DO11.10 mice. These cells were further stimulated with OVA_323–339 peptide (5 μg/ml) for 48 hours. Cell surface CD4, CD8, and OX40 expression were analyzed by flow cytometry. Representative plot of CD4, CD8, and OX40 expression from 3 independent studies.

Fig. 1

OVA induces OX40 expression primarily in CD4+ T cells in DO11.10 splenocytes. Splenocytes were isolated from DO11.10 mice. These cells were further stimulated with OVA_323–339 peptide (5 μg/ml) for 48 hours. Cell surface CD4, CD8, and OX40 expression were analyzed by flow cytometry. Representative plot of CD4, CD8, and OX40 expression from 3 independent studies.

Fig. 2

OX40 activating antibody induces CCL20 expression in DO11.10 splenocytes stimulated with OVA. The splenocytes were harvested from DO11.10 mice. These cells were further stimulated with OVA_323–339 peptide (5 μg/ml) and various concentrations of OX40 activating antibody for 48 hours, and CCL20 was examined by Western blot analysis. Representative image of CCL20 induction from 3 independent studies.

Fig. 3

Activated DO11.10 CD4+ cells express CCL20. DO11.10 splenocytes were cultured with and without OX40 activating antibody (4 μg/ml) in the presence of OVA_323–339 peptide (5 μg/ml) for 48 hours. CD4+ T lymphocytes were isolated using EasySep® Mouse CD4+ T Cell Enrichment Kit (StemCell Technologies). CCL20 in the CD4+ cells was further analyzed by Western blot. Note: OX40 activating antibody markedly increased the expression of CCL20 in CD4+ T cells. Representative image of CCL20 induction from 3 independent studies.

Fig. 4

NF-κB, JNK, and MEK inhibitors suppress OX40-mediated CCL20 expression in DO11.10 lymphocytes stimulated with OVA. The splenic lymphocytes were harvested from DO11.10 mice. These cells were further stimulated with OVA_323–339 peptide (5 μg/ml) in the presence or absence of OX40 activating antibody (4μg/ml) up to 72 hours, and CCL20 was examined by Western blot analysis. Representative image of CCL20 from 2 independent studies.

Fig. 5

PI3K, NF-κB, JNK, and MEK inhibitors suppress OX40-mediated IL-17A production in DO11.10 splenocytes stimulated with OVA. The splenocytes were harvested from DO11.10 mice. These cells were further stimulated with OVA_323–339 peptide (5 μg/ml) in the presence or absence of OX40 activating antibody (4μg/ml) up to 72 hours, and secreted IL-17A was examined by ELISA. Data represent the mean ± SD of 2 independent experiments (*p < 0.05; **p < 0.01).

Fig. 6

Intranasal inhalation of OVA and OX40 activating antibody-stimulated cell lysate significantly enhances antigen-induced airway inflammation in DO11.10 mice. DO11.10 splenocytes were primed with OVA alone or OVA plus OX40 activating antibody in vitro for 72 hours. Then, cell lysates derived from 5 × 10⁷ cells of each experimental group were administered into the lungs of recipient DO11.10 mice along with additional OVA or BSA (100 μg) via intranasal inhalation. Twenty four hours later, the lungs were collected for histological examination. Note: The cell lysate derived from the cells treated with OX40 activating antibody induced marked peribronchial and perivascular congregation of inflammatory cells, which was substantially attenuated by local treatment of CCL20 neutralizing antibody (n = 3 per group).

Fig. 7

Intranasal inhalation of cell lystate from OVA and OX40 activating antibody-stimulated cells significantly enhances Ccr6 transcript level in antigen-induced airway inflammation. DO11.10 splenocytes were stimulated with OVA alone or OVA plus OX40 activating antibody in vitro for 72 hours. Then, cell lysates derived from 5 × 10⁷ cells of each experimental group were administered into the lungs of recipient DO11.10 mice along with additional OVA or BSA (100 μg) via intranasal inhalation (n = 3 per group). Twenty four hours later, the lungs were harvested for total RNA isolation. The transcription of Ccr6 genes was measured by real time-PCR. Note: The cell lysate derived from the cells treated with OX40 activating antibody significantly enhanced Ccr6 signal in the antigen-challenged airway (*p < 0.05).