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

樹枝狀細胞(Dendritic cells, DC)是一種抗原呈現細胞 (antigen presenting cells, APC)，能將外來抗原吞噬，並將抗原呈現至細胞表面，刺激T細胞、B細胞與NK細胞的活化。近來有研究人員由犬之骨髓及週邊血中培養出DC，但對其表面抗原及功能特性皆未有進一步分析與探討。T細胞、B細胞與NK細胞皆受DC之調控，DC功能成熟度決定Th1或Th2細胞免疫之免疫抑制或增強反應，而DC的成熟度與腫瘤微環境有很微妙的關係為目前非常重要的研究。有關犬DC的研究方法所知甚少，我們由犬周邊血中成功培養DC，由犬周邊血中培養DC，其表面抗原經分析有CD1a+、CD11c+、CD14+、CD40+、CD86+及MHC II+之表現。其中人類CD40及CD80之單株抗體可交互作用於犬樹枝狀細胞表面抗原，而CD86及CD83則無法交互偵測。對於細胞功能分析方面，犬DC具有顯著之抗原吞噬及刺激異體T細胞增生功能。我們利用此犬DC進行後續研究及臨床治療。進一步我們觀察犬DC在LPS 及TNF-alpha刺激成熟後之細胞激素分泌概況。DC是身體內主要分泌細胞激素的細胞，而犬DC的細胞激素分泌概況至今尚無相關報告。由表面抗原分析及功能性測試證明在LPS及TNF-alpha刺激下DC會進入成熟期。以Real-time RT-PCR觀察DC之細胞激素基因表現，發現LPS刺激之DC會大量表現IL-1beta、IL-10、IL-12p40、IL-13及TNF-alpha，將此DC與淋巴球細胞共同培養時，發現此DC會引發T淋巴球分化至第一型幫助者T細胞；而TNF-alpha刺激之DC則是大量表現IL-2、IL-4、IL-12p40、IL-13、TNF-alpha、TGF-beta、IFN-gamma及MCP-2，再進一步將此DC與淋巴球細胞共同培養時，發現此DC會引發T淋巴球分化至第二型幫助者T細胞。因此，犬DC在不同刺激下會釋放不同之細胞激素進而影響先天及後天免疫反應。在腫瘤與DC的關係中，我們以犬傳染性花柳性腫瘤 (CTVT) 為模式，探討DC與該疾病的病理致病機轉的關連性。犬樹枝狀細胞與CTVT細胞培養上凊液共同培養後，以流式細胞儀觀察，發現CTVT細胞培養上凊液確會引起樹枝狀細胞的死亡，其可能之死亡途徑為自然凋亡。並降低表面抗原之表現及抗原吞噬的能力。我們發現腫瘤生長初期病犬主要免疫細胞-DC無論在功能上、數量上皆被顯著抑制，甚至引發DC死亡。在病犬身上抽取血液及淋巴結或是作樹枝狀細胞的體外培養皆發現存在相同的現象。在初步瞭解DC與腫瘤間之關係後，我們假設在腫瘤內以電基因細胞激素治療會改變腫瘤內微環境，使腫瘤更易於治癒。我們以小鼠動物模式試驗此假設。我們証明重複施以細胞激素電基因治療合併DC治療為十分有效之免疫治療方式，可使腫瘤內形成一種易於免疫攻擊之微環境。且此治療方式一旦改變腫瘤內微環境，還可進一步增加腫瘤對放射線之敏感度。在對腫瘤內微環境有一些瞭解後，我們在分析DC細胞激素分泌概況時，意外發現DC在發炎反應相關激素刺激下，會分泌CXCL7。而CXCL此類之蛋白與腫瘤微環境有十分密切之關連。由於DC 與CXCL7間的相互關係幾乎沒有任何報告，因此我們接著進一步分析這兩者之間的關係。首先將CXCL7以RT-PCR找出基因全長並放入表現載體，在轉染進入小鼠纖維母細胞3T3中，使其產生重組CXCL7蛋白。並進一步確認其有效之生物活性-促使嗜中性白血球細胞移動。我們發現DC在IL-1beta,IL-6, TGF-beta及TNF-alpha刺激下會有大量CXCL7表現並引發嗜中性白血球細胞移動，在IFN-gamma及LPS刺激下就無此現象。這表示在某些微環境下，DC會引發Th1細胞反應，反之則會引發Th2細胞反應，而CXCL7似乎與這些反應有些關連。我們亦發現CTVT在快速生長期會大量表現CXCL7，在腫瘤消退期則表現量下降。這結果顯示CXCL7對於促進腫瘤生長扮演一定的角色。綜上所言，在此論文中我們成功的建立了犬DC的體外培養方式，進一步分析其細胞激素分泌概況並發現CXCL7在DC與腫瘤間之特殊表現情形。此外，我們也証明在CTVT中DC功能會受到顯著的抑制。最後，我們也証明在腫瘤內以電基因細胞激素治療會改變腫瘤內微環境，使腫瘤更易於治癒並增加腫瘤對放射線之敏感度。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.定書 I謝 III要 Vbstract VIIontents XIcheme 1utline 2hapter 1. Background and Literatures review 3HE ROLE OF DENDRITIC CELLS IN IMMUNE SYSTEM 3ENDRITIC CELLS IN CANCER 4ANINE DENDRITIC CELLS CULTURE AND CHARACTERIZATION 5C AND CYTOKINES 6C AND CHEMOKINES 7ANINE TRANSMISSIBLE VENEREAL TUMOR 9C AND CTVT 10ICROENVIRONMENT AND TUMOR 10YPOTHESIS 12ESEARCH OBJECTIVES 15EFERENCE: 18hapter 2. Characterization of canine monocyte-derived dendritic cells with phenotypic and functional differentiation 25BSTRACT 25NTRODUCTION 27ATERIALS AND METHODS 28ource Animals and the Generation of Monocyte-Derived DC 28orphological Examination 29low Cytometry Analysis 30mmuno-precipitation and immuno-blot analysis. 30eal-Time RT-PCR 31easuring FITC-Dextran Uptake 33tatistical Analysis 34ESULTS 34eneration of Monocyte-derived DC 34orphology of Canine Monocyte-Derived DC 36C Phenotype 36ndocytotic Activity 37ixed-Lymphcyte Reaction 37PS-Dependent TNF-alpha Production Capacity Between the Monocytes iDC and mDC 38ISCUSSION 38EFERENCES 42IGURE LEGENDS 46ABLE 49ABLE 49IGURE 52hapter 3. Cytokine profiles of canine monocyte-derived dendritic cells as a function of lipopolysaccharide- or tumor ecrosis factor-alpha-induced maturation 57BSTRACT 57NTRODUCTION 59ATERIALS AND METHODS 61nimals and generation of DC 61low cytometry analysis of cell surface phenotypes 62xtraction of RNA from DC 62everse transcription of mRNA into cDNA 63rimers 63uantitative RT-PCR 63ata analysis of RT-PCR 64easuring FITC-dextran uptake 64ix Lymphocyte Reaction (MLR) 65llogeneic MLR and intracellular cytokine staining 65tatistics 66ESULTS 67eneration of canine monocyte-derived DCs 67henotypic and functional changes during maturation of canine DC 67ifferential cytokine expression by iDC, LPS-mDC, and TNF-alpha-mDC 68DC exhibited a Th1- or Th2-driving capacity after treatment with LPS or TNF-alpha 69ISCUSSION 71EFERENCE 76ABLE 81IGURE LEGENDS 83IGURE 85hapter 4. Transient downregulation of monocyte-derived dendritic-cell differentiation, function, and survival during tumoral rogression and regression in an in vivo canine model of transmissible venereal tumor 91BSTRACT 91NTRODUCTION 93ATERIALS AND METHODS 95xperimental animals, tumor inoculation, and determination of growth stage 95eneration of peripheral blood mononuclear cells, monocytes, and monocyte-derived DCs 95ample preparation of draining lymph nodes of CTVT dogs 96urification of tumor infiltrating lymphocytes (TIL) 97low cytometry 97eal-time RT-PCR analysis of surface markers CD80, CD83, and CD86 and cytokine IL-12 expression 98nzyme-linked immunosorbent assay of TNF-alpha 99ITC-dextran uptake 100ixed leukocyte reaction 100ffect of CTVT cell-culture supernatants on monocyte-derived DCs 101ffect of CTVT on monocytes 102ESULTS 102rowth phases 102hanges in numbers of PBMCs, monocytes, and DCs during tumoral growth 102xpression of surface markers on PBMCs, monocytes, iDCs, and mDCs 103C endocytic activity and allogeneic MLR results 103L-12 gene expression and TNF-alpha secretion by normal, P-phase, and R-phase DCs under lipopolysaccharide stimulation 104C killing by CTVT cell-culture supernatants 104ecreased number of DCs in the draining LN of CTVT dogs 105ISCUSSION 106EFERENCE: 110IGURE LEGENDS 115ABLE 117IGURE 120hapter 5. Synergistic Anti-Tumor Effect of Combination Radio- and Immuno-therapy by Electro-gene Therapy Plus Intra-tumor njection of Dendritic Cells 131BSTRACT 131NTRODUCTION 132ATERIALS AND METHODS 134nimal and tumor model 134ubcutaneous tumor model 134lasmids 134lectro-gene therapy protocol and transfer efficiency 135ultured murine dentritic cells 136mmunotherapy protocols 136mmunohistochemical (IHC) assay of EGT-treated tumors 137K cell activity assay 137ytokine release assay 138H-thymidine incorporation the T cell proliferation assay 139n vitro cytotoxicity assay (CTL) 139tatistical analyses 140ESULTS 141umor growth inhibition by combination of IL-2/GM-CSF EGT followed by DC treatment 141vidence of greater tumor infiltrating lymphocyte activation by the combination of IL-2/GM-CSF followed by DC treatment 141ncrease in NK cell activity by the combination of IL-2/GM-CSF EGT followed by DC treatment 141pecific T cell responses by the combination of IL-2/GM-CSF EGT followed by DC treatment 142ignificant enhancement of radiation by neoadjuvant immunotherapy with IL-2/GM-CSF EGT followed by DC treatment 142ystemic anti-tumor effect by the combination of IL-2/GM-CSF EGT followed by DC and radiation treatment 143ISCUSSION 143EFERENCES 146IGURE LEGENDS 149IGURE 152hapter 6. Cloning and expression of canine CXCL7 and its functional expression in dendritic cells under various maturation tage 159BSTRACT 159NTRODUCTION 161ATERIALS AND METHODS 163nimals and cells PBMC isolation and culture 163eneration of DC 163xtraction of RNA from PBMC and DC 164everse transcription of mRNA into cDNA 164T-PCR and RACE 165xpression of CXCL7 in BALB/3T3 cells 166rimers for Quantitative RT-PCR 166uantitative RT-PCR 167ata analysis 167roduction of rabbit antiserum against CXCL7 168estern blot analysis 168urface-Enhanced Laser Desorption /ionization Time-of-Flight Mass Spectrometry (SELDI-TOF-MS) assay 168hemotaxis assay 169tatistical analysis 171ESULTS 171olecular cloning and analysis of CXCL7 cDNA 171ukaryotic expression of CXCL7 172n vitro bioactivity of CXCL7 172he expression of CXCL7 in canine DC 173C attrack neutrophils under various stimulators 173ISCUSSION 174EFERENCE 178IGURE LENGEND 182IGURE 184hapter 7. CXCL7 overexpression is associated with the progression of canine transmissible venereal tumor (CTVT) 191BSTRACT 191NTRODUCTION: 192ATERIALS AND METHODS: 195n vivo tumor growth 195urification of CTVT cells and tumor infiltrating lymphocytes 195xtraction of RNA from CTVT 196everse transcription of mRNA into cDNA 196rimers for Real-time RT-PCR 196eal-time RT-PCR 197estern blot analysis 198tatistical analysis 198ESULTS: 199ssociation between CXCL7 overexpression and growth pattern 199 phase TIL inhibited the expression of CXCL7 in P phase CTVT cells. 199he expression of CXCL7 was regulated by IL-6 in CTVT cells. 199nhibition of expression of CXCL7 in CTVT cells 200ISCUSSION: 201EFERENCE: 204IGURE LEGEND 207IGURE 209hapter 8. Conclusion 21