The effect of downregulation of dendritic-cell function and CXCL7 expression in tumor microenvironmrnt during tumoral progression

• Main Authors: Yu-Shan Wang, 王愈善
• Other Authors: Rea-min Chu
• Format: Others
• Language: en_US
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/91246374770421351539

博士 === 國立臺灣大學 === 獸醫學研究所 === 96 === Dendritic cells (DC) play fundamental roles both in innate and adaptive immune responses and are also critical in fighting against tumor growth by presenting tumor-specific antigens to initiate an effective immune response. DC capture pathogens and to present antigens to T cells in lymph nodes, the organ for the generation of immunity and tolerance. DC are found in tumors in animals including human, dog and mice. Tumors could inhibit immunity, escape from immunosurveillance, especially in tumor microenvironment by many pathways. Due to low DC numbers in tissues naturally, the development of techniques to generate large numbers of DC in culture from either proliferating CD34+ progenitors or non-proliferating CD14+ monocytic precursors is essential for DC immunotherapy. In this thesis, we have developed a procedure that could efficiently generate canine DC from peripheral blood monoclear cells (PBMC) in a relatively large amount. Using real-time RT-PCR, the expression of CD80, CD83, and CD86 were compared between immatureDC (iDC) and mature DC (mDC), and the functional profiles of the monocyte-derived DC were also determined. These results facilitate the use of canine DC for further immunotherapy research and clinical application. To understand the relationship between DC and CTVT, we investigated the effects of CTVT on monocyte-derived canine DC. CTVT impair the differentiation of DC, inhibited antigen uptake and presentation, and caused apoptosis of monocytes and DC. During spontaneous regression, DC activity is substantially recovered. Reestablished DC are believed to be potentially important host factors that push the tumor toward to regression. We hypothesized that immunological favorable microenvironment can be restored by intratumoral electrogene cytokine therapy. We confirmed this hypothesis in both mice and canine model. We have demonstrated that repeated cytokine EGT followed by intratumoral DC injection is an effective immunotherapy protocol. Intratumoral injection of DC is critical for tumor infiltration lymphocyte activation. A cytokine rich microenviroment is essential for DC maturation. We also found a cytokine rich microenvironment is synergistic with radiation. All of those approach confirm the importance of immunotherapybased on dendritic cells. In the following step, the profiles of cytokines expressed during canine DC maturation were investigated. The DC were induced to express different sets of cytokines following stimulation by LPS or TNF-alpha. The DC stimulated by LPS released a set of cytokines that promoted Th1 activity, whereas DC stimulated by TNF-alpha produced a set of cytokines that promoted Th2 activity. Thus, in addition to the up-regulation of surface molecules, including MHC II, CD11c, CD80, CD83, and CD86, cytokine release profiles are another important component of DC activation directed to different effector functions. During the cytokine profile assay, we found that DC could express CXCL7 under proinflammatory cytokines stimulation. Because it is poor understood about how DC and CXCL7 interacted, we further assay the expression of CXCL7 in canine DC. Full length canine CXCL7 was cloned and expressed in BALB/3T3 cells. Migration assay confirmed the bioactivity of canine CXCL7. DC expressed significantly higher levels of CXCL7 gene under IL-1beta and TNF-alpha stimulating. We also demonstrated that canine CXCL7 expressed by BALB/3T3 cells could induce canine neutrophils recruitment and suggest that this chemokine play a similar role, as other species do, in leukocytes infiltration. Dog DC express CXCL7 during IL-1beta, IL-6, TGF-beta and TNF-alpha stimulating indicates that migration of neutrophils can be influenced by DC. Based on the finding we described above, we further clarified the role of CXCL7 and DC in CTVT model. We analyzed CXCL7 protein expression in CTVT and investigated the clinical implications. Strong CXCL7 expression correlated with higher tumor stage. CXCL7 overexpression was associated with progression phase of CTVT. Regression phase express very low level of CXCL7. These results suggest a role for CXCL7 in the clinical course of CTVT. In conclusion, we had established canine DC research and therapy model for dog cancer and found the importance of CXCL7 in DC immunosuppression.