Fibroblast and T cells conditioned media induce maturation dendritic cell and promote T helper immune response

Abstract

Dendritic cells (DCs) induce pathogen-specific T cell responses. We comprehensively studied the effects of addition of maturation stimulus, fibroblasts (fibroblast conditioned medium), PHA activated T cells (T cell conditioned medium), and mixture of fibroblast & PHA activated T cells (FCM-TCCM) conditioned media on maturation of DCs. Monocytes were cultured with GM-CSF and IL-4 for five days. Maturation factors included MCM and TNF-α as control group. FCM and TCCM, or FCM-TCCM supernatant were considered as the treatment group. Tumor antigens were added at day five. Matured DCs were harvested at day seven. Phenotypic and functional analyses were carried out using anti (CD14, CD80, CD86, CD83 and HLA-DR) monoclonal antibodies. Phagocytic activity, mixed lymphocyte reaction (MLR) and cytokine production were also evaluated. At the end of culturing period, significantly fully matured DCs with large amount cytoplasm and copious dendritic projections were found in the presence of MCM, TNF-α with or without FCM, TCCM, FCM as well as TCCM. Flow cytometric analysis revealed that expression of CD14 decreased in particular in treated DCs, at the 5(th) day and expression of CD80, CD86 and HLA-DR was higher when FCM, TCCM, FCM plus TCCM were added to maturation factor. This study demonstrated that DCs matured with these methods had optimum function in comparison with either factor alone.

Keywords: Dendritic cell; Fibroblast; T cell.

Fig. 1

Monocyte-derived DCs. Control: DCs differentiated in the presence of MCM, TNF-α. FCM: DCs differentiated in the presence of MCM, TNF-α, FCM. TCCM: DCs differentiated in the presence of MCM, TNF-α, TCCM. FCM-TCCM: DCs differentiated in the presence of MCM, TNF-α, FCM-TCCM. The most of mature DCs were appeared as a single cells or loosely adherent aggregates reviewing by light microscopy. (400×).

Fig. 2

Flow cytometreic analysis of expression of CD14, CD80, CD86, HLA-DR and CD83. Monocyte-derived DCs differentiated in the presence of MCM, TNF-α, with or without FCM, TCM, FCM plus TCCM were harvested on day 7 and analyzed by using respective monoclonal antibodies and isotype controls. Data represent the mean ± SD of three independent experiments

Fig. 3

Changes in surface-antigen phenotype of DC treated with FCM, TCCM and FCM+TCCM. Human peripheral blood monocytes were cultured with GM-CSF and IL-4 for 7 d and further with medium alone (first), FCM (second), the TCCM of PHA- activated T cells (third), FCM+TCCM (forth). DC were examined for surface expression of HLA-DR, CD86, CD83, CD80 and CD14 by flow cytometry. Incorporation of FITC-conjugated dextran (F-DEX) was also assessed by flow cytometry.

Fig. 4

DC phagocytic activity. Phagocytic activity of MCM, TNF-α, with or without FCM, TCM, FCM plus TCCM treated immature and mature DCs was measured using FITC-conjugated bead uptake and results of percent and mean fluorescent intensity (MFI) of phagocytic cells. Data represent mean ± SD of three independent experiments. * indicates significant differences at P < 0.05.

Fig. 5

Cytokine release from allogenic MLR. Supernatants of MCM, TNF-α, with or without FCM, TCM, FCM plus TCCM matured DCs as well as of allogenic MLR, IFN-γ and IL-4 cytokines assay, respectively. The results are expressed as a mean of triplicates

Fig. 6

Cytokine release from DCs. Supernatants of MCM, TNF-α, with or without FCM, TCM, FCM plus TCCM matured DCs as well as supernatants were subjected to IL-12, IL-10cytokines assay respectively. The results are expressed as the mean of triplicates.

Fig. 7

Cytokine release from Allogenic MLR. Supernatants of MCM, TNF-α, with or without FCM, TCM, FCM plus TCCM matured DCs as well as of allogenic MLR IFN-γ and IL-4 cytokines assay respectively. The results are expressed as the mean of triplicates.