In vitro maturation and migration of immature dendritic cells after chemokine receptor 7 transfection

Abstract

Dendritic cells are specialized antigen-presenting cells that regulate immunity and tolerance. Chemokine receptor 7 (CCR7), which is expressed by mature dendritic cells, mediates the migration of the cells to secondary lymphoid organs and thus regulates immune responses. It has been demonstrated that immature dendritic cells can induce immune tolerance, but they do not express CCR7 and cannot migrate to secondary lymphoid organs. We transfected immature dendritic cells with a recombinant adenovirus carrying the CCR7 gene to obtain immature dendritic cells with the ability to migrate. The maturity of the cells was monitored by scanning electron microscopy and flow cytometry. In addition, we assessed the ability of cells to migrate and the function of the cells using in vitro chemotactic and mixed leukocyte reaction assays. The results showed that immature dendritic cells became semi-mature, exhibiting a mild upregulation of co-stimulatory molecular expression and a few dendritic processes. Immunofluorescence assay and Western blotting indicated that CCR7 protein expression increased significantly in immature dendritic cells following CCR7 gene transfection. The in vitro chemotactic assay showed a significantly enhanced ability to migrate in response to CCL19 following CCR7 gene transfection. Moreover, transfected cells showed an enhanced ability to stimulate allogeneic T cell proliferation in vitro, but their ability was significantly weaker than that of mature dendritic cells. Interleukin-10 inhibited the differentiation and maturation of immature dendritic cells. It is concluded that, following CCR7 gene transfection, immature dendritic cells exhibit an enhanced ability to migrate and some of the characteristics of mature cells. Thus, these cells are of potential clinical significance in studies of immune tolerance induction during skin grafting after severe burns.

Suppression of lentivirus-mediated transgenic dendritic cells in graft-versus-host disease after allogeneic bone marrow transplantation in mice

“…Flow cytometry revealed no expression of CCR7 in the pXZ9-imDC group, while there was CCR7 expression in the CCR7-imDC group. Moreover the expression level of CCR7 was higher in the immature DC group than in the mature DC group, and the expression of CD80, CD86, CD11c, and MHC-Ⅱ molecules was quite low in immature DCs transfected with the Ccr7 gene, suggesting that Ccr7 had been successfully introduced into the imDCs, which led to effective CCR7 protein expression (Xin et al, 2009). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to detect the impact of DCs on T cell proliferation in each group, and Transwell chamber culture was used to detect the in vitro chemotaxis of cells in each group.…”

“…The lentivirus that carried the Ccr7 gene and green fluorescent protein were constructed and preserved in our laboratory (Xin et al, 2009;Cao et al, 2010). DCs infected by the target gene-carrying virus and the empty virus were designated as CCR7-imDCs and pXZ9-imDCs, respectively.…”

“…Then mouse imDCs were prepared by referring to the method described in the literature (Xin et al, 2009;Wang et al, 2012). The virus carrying the Ccr7 gene (pXZ9-Ccr7) and the empty virus control (pXZ9) were added to the different groups of imDCs for transfection, and the corresponding transfected imDCs were designated CCR7-imDC and pXZ9-imDC, respectively.…”

“…In another study, it was shown that bone marrow-derived DCs did not migrate to the lymph nodes in Ccr7 gene knockout (Ccr7 -/-) mice (Hintzen et al, 2006). However, since imDCs lack CCR7 expression (Xin et al, 2009), their ability to migrate to the lymph nodes is weaker, which causes them to develop into mature DCs with little inhibition effect on T lymphocyte proliferation during their long stay in non-T cell zones. Therefore, by constructing modified imDCs that express chemokine receptors on their surface, which have a strong homing capacity for the draining and central lymph nodes, there is a great probability that their ability to induce immune tolerance may be enhanced.…”

“…Therefore, by constructing modified imDCs that express chemokine receptors on their surface, which have a strong homing capacity for the draining and central lymph nodes, there is a great probability that their ability to induce immune tolerance may be enhanced. In early experiments, our research team successfully constructed lentivirus vectors carrying the mouse Ccr7 gene (labeled with green fluorescence), namely, LV-CCR7, and obtained imDCs with high expression of CCR7 by infecting imDCs with the above-mentioned virus vector (Xin et al, 2009;Cao et al, 2010). Therefore, the aim of our present study was to investigate the impact of Ccr7-modified imDCs on GVHD after allo-HSCT and the mechanisms involved, so as to provide evidence for the prevention and treatment of GVHD in clinical practice. )…”