肿瘤微环境诱导不成熟树突状细胞为调节性树突状细胞的研究
发布时间：2018-07-27 10:23
【摘要】： 树突状细胞(Dendritic cell，DC)是目前发现的机体内功能最强大的抗原呈递细胞(Antigen-presenting cells，APC)，其表面的MHC类分子和MHC—肽复合物含量是其它APC的10～100倍。DC可有效地诱导初始T细胞(Naive T cell)增殖和应答，促进细胞毒性T淋巴细胞(Cytotoxic T lymphocytes，CTL)和辅助性T细胞(T help cells，Th)的生成，可见DC是机体免疫反应的启动者和参与者。由DC激活的T细胞介导的抗肿瘤免疫反应是机体抗肿瘤免疫的主导力量。有报道称，许多肿瘤组织中浸润有树突状样或表达DC表面标志的细胞，但尽管肿瘤组织内存在这类细胞，肿瘤细胞还是会逃脱免疫系统的监视而过度生长，甚至发生浸润和转移。因此，有关肿瘤免疫逃逸机制的研究是当前生物医学的一个前沿热点课题。目前，对于肿瘤细胞逃避DC对其表面抗原的提呈而发生免疫逃逸的机制尚未完全清楚。诸多研究指出，其机制可能是肿瘤细胞释放多种抑制性的细胞因子如VEGF、TGF-β、IL-10、M-CSF、PGE2等使DC成熟和分化发生障碍，抑制了DC抗原提呈能力并减弱其激活T细胞的能力，使这些DC对活化信号表现出低反应性。随着DC研究的不断深入，各种DC亚群包括具有负向免疫调控作用的调节性DC的发现，肿瘤浸润DC的功能也变得更为复杂。肿瘤浸润DC中是否也存在着具有抑制功能的DC亚群昵?是否该群DC不但不具备免疫监视和免疫防御功能，反而会促进肿瘤细胞的生长和转移呢?基于这样的考虑，我们利用新鲜分离的肿瘤细胞模拟肿瘤微环境与体外培养的DC共孵育，研究了肿瘤微环境对DC的表型和功能特征的影响，以期进一步了解肿瘤微环境对DC的影响以及肿瘤浸润DC对肿瘤生长和转移的作用，为肿瘤免疫逃逸的机制研究增加新的认识。 首先，我们选用小鼠肺癌细胞株3LL皮下接种C57BL／6小鼠，复制荷瘤小鼠模型，分离合适大小的肿瘤组织，用胶原酶消化后碾磨成单细胞悬液，让其贴壁生长。一周后即可得到形态相似的，能够生长的原代肿瘤细胞。新鲜分离的肿瘤细胞比肿瘤细胞系具有更好的性状，更能模拟体内的肿瘤微环境。我们制备了GM-CSF和IL-4培养了5天的小鼠骨髓来源的CD11c~+DC，此时CD11c~+DC细胞表面Ia、CD40、CD80、CD86的水平都比较低，具有较强的吞噬功能，可以认为是一种不成熟DC。我们用新鲜分离的原代肿瘤细胞与不成熟DC体外共培养，用来模拟肿瘤微环境与DC的相互作用，开展以下实验研究。 对于肿瘤周围浸润的DC的状态目前还存在着争议，但大量数据表明，肿瘤细胞通过分泌一些免疫抑制性细胞因子阻碍其周围浸润的DC成熟使之处于不成熟状态；为了阐明肿瘤周围浸润DC的状态，我们利用transwell系统用肿瘤培养上清对体外培养的不成熟DC和成熟DC分别进行趋化，结果发现肿瘤培养上清能够趋化大量不成熟DC，几乎不趋化成熟DC。因此，我们将体外培养5天的不成熟DC与新鲜分离制备的肿瘤细胞共培养60h，之后用CD11c~+磁珠分离DC，检测了诱导后(与肿瘤共培养后)的DC的表型及其在LPS刺激后的表型变化，并将之与诱导前的不成熟DC、不成熟DC加LPS刺激后活化成熟的DC进行了比较。结果发现，与成熟DC相比，肿瘤细胞诱导后的DC表面分子CD86、Ia、CD11c下调，CD11b上调，而CD40、CD80的变化不大，并且在LPS刺激后表面MHCⅡ类分子和共刺激分子并没有显著升高，表现为CD11c~(low)Ia~(low)CD11b~(high)的稳定的表型特征。 吞噬功能在一定程度上代表了DC的成熟度和功能状态，一般认为DC越成熟吞噬能力越弱，而抗原递呈能力越强。我们进一步分析了肿瘤细胞诱导形成的CD11c~(l0w)Ia_(low)CD11b~(high)DC的吞噬功能，结果发现该群CD11c~(low)Ia~(low)CD11b~(high)DC与不成熟DC相似，具有很强的吞噬能力，甚至强于不成熟DC的吞噬能力，即使在LPS刺激后，其仍然显示很高的吞噬能力，但不成熟DC在LPS刺激后变成了成熟DC，其吞噬能力显著下降。提示肿瘤细胞体外诱导形成的CD11c~(low)Ia~(low)CD11b~(high)DC不同于不成熟DC和成熟DC，并处于功能稳定状态。 从细胞因子分泌谱看，CD11c~(low)Ia~(low)CD11b~(high)DC自发分泌高量的IL-10、NO、PGE2，而IL-12的分泌量很低，LPS刺激后变化不大，进一步提示该CD11c~(low)Ia~(low)CD11b~(high)DC处于稳定的状态并且可能具有下调免疫应答的能力。 DC的重要功能是抗原递呈能力，为了探讨CD11c~(low)Ia~(low)CD11b~(high)DC的抗原递呈能力，我们将CD11c~(low)Ia~(low)CD11b~(high)DC与OVA_(323-339)抗原肽特异性反应的CD4~+T细胞在OVA_(323-339)抗原肽存在的条件下共培养，结果发现CD11c~(low)Ia~(low)CD11b~(high)DC刺激抗原肽特异性T细胞的能力显著的低于成熟DC。CD11c~(low)Ia~(low)CD11b~(high)DC刺激抗原肽特异性T细胞的能力很弱，表明其本身的抗原提呈功能很弱。我们检测了培养上清及T细胞胞内细胞因子的分泌情况，结果显示CD11c~(low)Ia~(low)CD11b~(high)DC仍然能够刺激CD4~+T细胞分泌一定水平的IL-2和IFN-γ。 鉴于CD11c~(low)Ia~(low)CD11b~(high)DC的以上特点，我们推测该细胞可能具有负向免疫调节作用。我们将CD11c~(low)Ia~(low)CD11b~(high)DC加入到OVA_(323-339)抗原肽特异性CD4~+T／maDC／OVA_(323-339)的共培养体系中，结果发现CD11c~(low)Ia~(low)CD11b~(high)DC有效地抑制了T细胞的增殖，同样检测了T细胞胞内细胞因子的分泌情况，结果表明，CD11c~(low)Ia~(low)CD11b~(high)DC仍然能够刺激T细胞分泌一定水平的IL-2和IFN-γ。因此我们将肿瘤细胞诱导形成的这群CD11c~(low)Ia~(low)CD11b~(high)Dc称为调节性DC(regulatory DC，DCreg)。 为了明确我们在体外共培养体系中诱导得到的调节性DC是否在肿瘤瘤体内存在相应的DC亚群，我们根据该调节性DC的表型特点，分析了肿瘤单个核细胞中CD11c~+细胞群。该调节性DC最重要的特点就是高表达CD11b、低表达CD11c及Ia，因此我们用CD11b-APC、CD11c-FITC及Ia-PE三色标记肿瘤单个核细胞中CD11c~+细胞群，FACS分析发现这群细胞中明显存在一群CD11b~(high)CD11c~(low)细胞，进一步分析CD11b~(high)CD11c~(low)细胞的Ia的表达，我们发现存在低、中、高连续表达的三群细胞。关于具体哪群或哪几群细胞具有与体外肿瘤细胞共培养体系所诱导形成的调节性DC具有相似的功能特征，我们打算用FACSVantage将这三群细胞分选出来，分析它们的细胞因子分泌情况和抑制功能，最终来证实肿瘤微环境内确实存在一群表型和功能与我们通过体外肿瘤细胞共培养体系所诱导产生的DCreg相似的肿瘤浸润DC亚群。 以上研究表明，肿瘤微环境确实能够诱导不成熟DC向DCreg的分化，，那么，肿瘤细胞以何种形式诱导了不成熟DC向DCreg的分化呢?是肿瘤细胞分泌的免疫抑制性可溶性分子，还是肿瘤细胞表面表达的抑制性膜分子，抑或是两者共同发挥了作用?首先，我们用半定量RT-PCR检测发现，3LL肿瘤细胞高表达M-CSF、TGF-β、VEGF等具有免疫抑制作用的细胞因子。为了明确3LL肿瘤细胞以何种形式诱导了不成熟DC向DCreg的分化，我们选择了Transwell系统进一步开展了研究。虽然多聚甲醛固定肿瘤细胞可以阻断肿瘤细胞可溶性因子的释放，可以用来研究肿瘤表面膜分子的作用，但由于不成熟DC具有很强的吞噬作用，固定肿瘤细胞会被不成熟DC吞噬，因此我们放弃了这种研究模式，采用了Transwell系统，Transwell系统可以将两种细胞有效的隔开，避免了细胞与细胞的直接接触，可用来研究肿瘤细胞分泌的可溶性因子的作用。我们将不成熟DC直接加入预先铺有肿瘤细胞的24孔板中或加入到肿瘤细胞上方的Transwell小室中，比较了两者细胞的抑制功能，结果发现，两组处理的DC都能有效地抑制T细胞的增殖，且抑制的水平没有显著性差异，表明肿瘤细胞分泌的可溶性因子就足以使不成熟DC向DCreg转化而具备了抑制功能。进一步阐明哪种或哪些可溶性因子参与了不成熟DC向DCreg转化，我们将采用中和性抗体和／或抑制剂进行阻断和联合阻断实验。 肿瘤分泌的免疫抑制性可溶性因子构成的肿瘤微环境，可以诱导进入肿瘤的不成熟DC转化为调节性DC，这群DC高分泌IL-10、PGE2和NO，在体外可以明显抑制抗原特异性的CD4~+T细胞的增殖，我们推测，该群DC是否不但不具备免疫监视和免疫防御功能，反而能够促进肿瘤细胞的生长和转移呢?为了探讨CD11c~(low)Ia~(low)CD11b~(high)DC是否促进肿瘤的转移，我们建立了3LL肿瘤的肺转移模型，我们先用不成熟DC或DCreg尾静脉注射小鼠，24小时后再尾静脉注射小鼠3LL肺癌细胞，20天后处死小鼠，用墨汁灌注的方法观察肺部的肿瘤转移灶，结果显示，注射DCreg组的转移灶多于不成熟DC组。提示了该群CD11c~(low)Ia~(low)CD11b~(high)DC不但不具备免疫监视和免疫防御功能，反而促进肿瘤细胞的转移。 综上所述，肿瘤细胞可以通过组成性和／或诱导性分泌一些趋化因子，招引不成熟DC进入瘤体，不成熟DC在肿瘤细胞释放的可溶性因子作用下，进一步分化为高分泌IL-10、PGE2和NO但低分泌IL-12的CD11c~(low)Ia~(low)CD11b~(high)调节性DC，该调节性DC能在体外抑制抗原肽特异性CD4~+T细胞的增殖反应，并且可以在体内促进肿瘤转移。本研究还初步证明了荷瘤小鼠瘤体内可能存在相应的调节性DC亚群，该实验结果为肿瘤免疫逃逸提供了又一新的机制和解释。
[Abstract]:Dendritic cell (DC) is the most powerful antigen presenting cell (Antigen-presenting cells, APC) that has been found in the body. The content of MHC class and MHC peptide complex on the surface is 10~100 times that of other APC, which can effectively induce the proliferation and response of the initial T cells (Naive) and promote the cytotoxicity of lymphoid cells. The formation of Cytotoxic T lymphocytes (CTL) and auxiliary T cells (T help cells, Th) can be seen that DC is the promoter and participant of the body's immune response. The anti-tumor immune response mediated by DC activated T cells is the dominant force in the body's anti-tumor immunity. It is reported that a dendritic like or expression surface mark is infiltrated in a number of tumor tissues. However, although the tumor cells are in this kind of cells, the tumor cells will escape the surveillance of the immune system and be overgrown and even infiltrate and metastases. Therefore, the research on the immune escape mechanism of the tumor is a hot topic in the current biomedicine. The mechanism of immune escape is not completely clear. Many studies suggest that the mechanism may be that tumor cells release a variety of inhibitory cytokines such as VEGF, TGF- beta, IL-10, M-CSF, PGE2 and so on, which cause the maturation and differentiation of DC, inhibit the ability of DC antigen presenting and weaken its ability to activate T cells, and make these DC activated signals It shows low responsiveness. As DC studies continue to deepen, various DC subgroups include the discovery of a regulatory DC with negative immunoregulation, and the function of tumor infiltrating DC becomes more complex. Is there a subgroup of DC with inhibitory function in the tumor infiltrating DC? Whether the group of DC does not possess not only immune surveillance and immune defense work, but also the group of DC On this basis, we use fresh isolated tumor cells to simulate the tumor microenvironment and the DC co incubation in vitro. The effect of tumor microenvironment on the phenotypic and functional characteristics of DC is studied to further understand the effect of tumor microenvironment on DC and the invasion of DC. The role of tumor growth and metastasis has increased the new understanding of the mechanism of tumor immune escape.
First, we selected the mouse lung cancer cell line 3LL to inoculate C57BL / 6 mice subcutaneously, replicate the tumor bearing mice model, separate the appropriate size of the tumor tissue, and grind it into a single cell suspension with collagenase, let it grow on the wall. One week later, the tumor cells with similar morphology and long growth can be obtained. The fresh isolated tumor cells are more than the swelling. The tumor cell line has a better character and can mimic the microenvironment of the tumor in the body. We have prepared GM-CSF and IL-4 for 5 days of mouse bone marrow origin CD11c~+DC. At this time, the level of Ia, CD40, CD80, CD86 on the surface of CD11c~+DC cells is relatively low and has strong phagocytosis, which can be considered as a kind of immature DC. we use fresh separation. The primary tumor cells were co cultured with immature DC in vitro to simulate the interaction between tumor microenvironment and DC.
There is still controversy about the state of DC infiltrating around the tumor, but a lot of data show that the tumor cells are immaturity by secreting some immunosuppressive cytokines that inhibit the maturation of the surrounding DC, and to clarify the state of infiltration of DC around the tumor, we use the Transwell system to use the tumor culture supernatant. The immature DC and mature DC cultured in vitro were chemotactic respectively. The results showed that the tumor culture supernatant could chemotactic a large number of immature DC and hardly chemotaxis mature DC.. Therefore, we cultured the immature DC in vitro for 5 days with the fresh isolated tumor cells to co culture 60H, then the CD11c~+ magnetic beads were used to separate DC. The phenotype of the cultured DC and its phenotypic changes after LPS stimulation were compared with the immature DC, the immature DC plus the LPS stimulation of the mature DC. The results showed that, compared with the mature DC, the surface molecules of the DC surface, which were induced by the tumor cells, were in CD86, Ia, CD11c, and up. After stimulation, the surface MHC class II molecules and costimulatory molecules did not increase significantly, showing stable phenotypic characteristics of CD11c~ (low) Ia~ (low) CD11b~ (high).
Phagocytosis, to a certain extent, represents the maturity and functional state of DC. It is generally believed that the more mature the phagocytosis of DC is, the stronger the antigen presenting ability. We further analyzed the phagocytic function of CD11c~ (L0W) Ia_ (low) CD11b~ (high) DC induced by tumor cells. Mature DC has a strong phagocytosis and even stronger than the phagocytic ability of immature DC. Even after LPS stimulation, it still shows high phagocytosis, but immature DC becomes mature DC after LPS stimulation, and its phagocytic ability decreases significantly. It suggests that CD11c~ (low) Ia~ (low) CD11b~ (high), induced by tumor cells in vitro, is different from that of no Mature DC and mature DC, and are in a stable state of function.
From the cytokine secretion spectrum, CD11c~ (low) Ia~ (low) CD11b~ (high) DC spontaneously secretes a high amount of IL-10, NO, PGE2, but the IL-12 secretion is very low, and the LPS stimulus does not change very much, further suggesting that the DC is in a stable state and may have the ability to reduce the immune response.
The important function of DC is antigen presenting ability. In order to explore the antigen presenting ability of CD11c~ (low) Ia~ (low) CD11b~ (high) DC, we co culture the CD11c~ (low) Ia~ (low) Ia~ (low) and antigen peptide specific peptide in the presence of the antigen peptide (323-339). The ability of DC to stimulate the antigenic peptide specific T cells was significantly lower than that of the mature DC.CD11c~ (low) Ia~ (low) CD11b~ (high) DC stimulating antigen peptide specific T cells, which showed that the antigen presenting function was weak. IgH) DC can still stimulate CD4~+T cells to secrete a certain level of IL-2 and IFN- gamma.
In view of the above characteristics of CD11c~ (low) Ia~ (low) CD11b~ (high) DC, we speculate that the cell may have a negative immunomodulatory function. We add CD11c~ (low) Ia~ (low) CD11b~ (low) to the co culture system of 323-339 antigen specific peptide specificity / 323-339. The proliferation of T cells was inhibited and the secretion of intracellular cytokines in T cells was also detected. The results showed that CD11c~ (low) Ia~ (low) CD11b~ (high) DC still stimulated T cells to secrete a certain level of IL-2 and IFN- gamma. Y DC, DCreg).
In order to determine whether the regulatory DC induced by our in vitro co culture system has a corresponding DC subgroup in the tumor tumor, we analyze the CD11c~+ cell group in the tumor mononuclear cells based on the phenotypic characteristics of this regulatory DC. The most important characteristic of this regulatory DC is the high expression of CD11b, the low expression of CD11c and Ia, so we use it. CD11b-APC, CD11c-FITC and Ia-PE tricolor labeled CD11c~+ cell groups in tumor mononuclear cells. FACS analysis found a group of CD11b~ (high) CD11c~ (low) cells in this group. We further analyzed the expression of CD11b~ (high) CD11c~ cells. We found three groups of cells with low, medium and high continuous expression. Which group of cells have the similar functional characteristics with the regulatory DC induced by coculture system in vitro, we intend to use FACSVantage to separate these three groups of cells, analyze their cytokine secretion and inhibitory function, and finally confirm that there is a group of phenotypes and functions in the tumor microring. Similar tumor infiltrates into DC subgroup induced by co culture system of tumor cells in vitro. DCreg
The above studies have shown that the tumor microenvironment can indeed induce the differentiation of immature DC to DCreg, so in what form does the tumor cells induce the differentiation of immature DC to DCreg, the immunosuppressive soluble molecules secreted by the tumor cells, or the inhibitory membrane molecules expressed on the surface of the tumor cells, or both of them play a role together. First, we found that 3LL tumor cells express M-CSF, TGF- beta, VEGF and other cytokines with immunosuppressive effects by semi quantitative RT-PCR detection. In order to identify the form of 3LL tumor cells to induce the differentiation of immature DC to DCreg, we chose the Transwell system to carry out a study. Cell can block the release of soluble factors of tumor cells and can be used to study the role of tumor surface mask molecules. But because immature DC has a strong phagocytosis, the fixed tumor cells will be phagocytic by immature DC. Therefore, we abandoned this research pattern, using the Transwell system, and the Transwell system can make two kinds of cells effective. Isolation, avoiding direct contact between cells and cells, can be used to study the role of soluble factors secreted by tumor cells. We add immature DC directly into 24 foramen of cancer cells or add to the Transwell cells above the tumor cells and compare the inhibitory function of the two cells. The result is that the two groups of D are treated. C can effectively inhibit the proliferation of T cells, and there is no significant difference in the level of inhibition, indicating that the soluble factors secreted by the tumor cells are sufficient to inhibit the transformation of immature DC to DCreg. Which or which kinds of soluble factors are involved in the transformation of immature DC to DCreg, we will use neutralizing antibodies and / / Blocking and joint blocking experiments were performed or depressant.
The tumor microenvironment, which is secreted by the immunosuppressive soluble factors, can induce the immature DC into the tumor to convert into regulatory DC. This group of DC hypersecreting IL-10, PGE2 and NO can obviously inhibit the proliferation of antigen specific CD4~+T cells in vitro. We speculate whether this group DC not only does not have immune surveillance and immune defense. In order to investigate whether CD11c~ (low) Ia~ (low) CD11b~ (high) DC promotes tumor metastasis, we have established a lung metastasis model of 3LL tumor. We first injected the immature DC or DCreg tail vein to mice, and then injected the mouse 3LL lung cancer cells into the tail vein in 24 small hours, and then killed the mice after 20 days. The tumor metastasis of lung was observed with the method of ink infusion. The results showed that the metastasis of DCreg group was more than that of the immature DC group. It suggested that the group CD11c~ (low) Ia~ (low) CD11b~ (high) DC not only did not have immune surveillance and immune defense, but promoted the metastasis of tumor cells.
To sum up, the tumor cells can induce immature DC into the tumor through the composition and / or inducible secretion of some chemokines. Under the action of the soluble factors released by the tumor cells, the immature DC further differentiates into the CD11c~ (low) Ia~ (low) CD11b~ (high) regulatory IL-10, PGE2 and NO, but the low secretion of IL-12. It can inhibit the proliferation of antigenic peptide specific CD4~+T cells in vitro, and can promote tumor metastasis in vivo. This study also preliminarily demonstrated that there may be a corresponding regulatory DC subgroup in tumor bearing mice, which provides another new mechanism and explanation for tumor immune escape.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392;R730.3
本文编号：2147524
[Abstract]:Dendritic cell (DC) is the most powerful antigen presenting cell (Antigen-presenting cells, APC) that has been found in the body. The content of MHC class and MHC peptide complex on the surface is 10~100 times that of other APC, which can effectively induce the proliferation and response of the initial T cells (Naive) and promote the cytotoxicity of lymphoid cells. The formation of Cytotoxic T lymphocytes (CTL) and auxiliary T cells (T help cells, Th) can be seen that DC is the promoter and participant of the body's immune response. The anti-tumor immune response mediated by DC activated T cells is the dominant force in the body's anti-tumor immunity. It is reported that a dendritic like or expression surface mark is infiltrated in a number of tumor tissues. However, although the tumor cells are in this kind of cells, the tumor cells will escape the surveillance of the immune system and be overgrown and even infiltrate and metastases. Therefore, the research on the immune escape mechanism of the tumor is a hot topic in the current biomedicine. The mechanism of immune escape is not completely clear. Many studies suggest that the mechanism may be that tumor cells release a variety of inhibitory cytokines such as VEGF, TGF- beta, IL-10, M-CSF, PGE2 and so on, which cause the maturation and differentiation of DC, inhibit the ability of DC antigen presenting and weaken its ability to activate T cells, and make these DC activated signals It shows low responsiveness. As DC studies continue to deepen, various DC subgroups include the discovery of a regulatory DC with negative immunoregulation, and the function of tumor infiltrating DC becomes more complex. Is there a subgroup of DC with inhibitory function in the tumor infiltrating DC? Whether the group of DC does not possess not only immune surveillance and immune defense work, but also the group of DC On this basis, we use fresh isolated tumor cells to simulate the tumor microenvironment and the DC co incubation in vitro. The effect of tumor microenvironment on the phenotypic and functional characteristics of DC is studied to further understand the effect of tumor microenvironment on DC and the invasion of DC. The role of tumor growth and metastasis has increased the new understanding of the mechanism of tumor immune escape.
First, we selected the mouse lung cancer cell line 3LL to inoculate C57BL / 6 mice subcutaneously, replicate the tumor bearing mice model, separate the appropriate size of the tumor tissue, and grind it into a single cell suspension with collagenase, let it grow on the wall. One week later, the tumor cells with similar morphology and long growth can be obtained. The fresh isolated tumor cells are more than the swelling. The tumor cell line has a better character and can mimic the microenvironment of the tumor in the body. We have prepared GM-CSF and IL-4 for 5 days of mouse bone marrow origin CD11c~+DC. At this time, the level of Ia, CD40, CD80, CD86 on the surface of CD11c~+DC cells is relatively low and has strong phagocytosis, which can be considered as a kind of immature DC. we use fresh separation. The primary tumor cells were co cultured with immature DC in vitro to simulate the interaction between tumor microenvironment and DC.
There is still controversy about the state of DC infiltrating around the tumor, but a lot of data show that the tumor cells are immaturity by secreting some immunosuppressive cytokines that inhibit the maturation of the surrounding DC, and to clarify the state of infiltration of DC around the tumor, we use the Transwell system to use the tumor culture supernatant. The immature DC and mature DC cultured in vitro were chemotactic respectively. The results showed that the tumor culture supernatant could chemotactic a large number of immature DC and hardly chemotaxis mature DC.. Therefore, we cultured the immature DC in vitro for 5 days with the fresh isolated tumor cells to co culture 60H, then the CD11c~+ magnetic beads were used to separate DC. The phenotype of the cultured DC and its phenotypic changes after LPS stimulation were compared with the immature DC, the immature DC plus the LPS stimulation of the mature DC. The results showed that, compared with the mature DC, the surface molecules of the DC surface, which were induced by the tumor cells, were in CD86, Ia, CD11c, and up. After stimulation, the surface MHC class II molecules and costimulatory molecules did not increase significantly, showing stable phenotypic characteristics of CD11c~ (low) Ia~ (low) CD11b~ (high).
Phagocytosis, to a certain extent, represents the maturity and functional state of DC. It is generally believed that the more mature the phagocytosis of DC is, the stronger the antigen presenting ability. We further analyzed the phagocytic function of CD11c~ (L0W) Ia_ (low) CD11b~ (high) DC induced by tumor cells. Mature DC has a strong phagocytosis and even stronger than the phagocytic ability of immature DC. Even after LPS stimulation, it still shows high phagocytosis, but immature DC becomes mature DC after LPS stimulation, and its phagocytic ability decreases significantly. It suggests that CD11c~ (low) Ia~ (low) CD11b~ (high), induced by tumor cells in vitro, is different from that of no Mature DC and mature DC, and are in a stable state of function.
From the cytokine secretion spectrum, CD11c~ (low) Ia~ (low) CD11b~ (high) DC spontaneously secretes a high amount of IL-10, NO, PGE2, but the IL-12 secretion is very low, and the LPS stimulus does not change very much, further suggesting that the DC is in a stable state and may have the ability to reduce the immune response.
The important function of DC is antigen presenting ability. In order to explore the antigen presenting ability of CD11c~ (low) Ia~ (low) CD11b~ (high) DC, we co culture the CD11c~ (low) Ia~ (low) Ia~ (low) and antigen peptide specific peptide in the presence of the antigen peptide (323-339). The ability of DC to stimulate the antigenic peptide specific T cells was significantly lower than that of the mature DC.CD11c~ (low) Ia~ (low) CD11b~ (high) DC stimulating antigen peptide specific T cells, which showed that the antigen presenting function was weak. IgH) DC can still stimulate CD4~+T cells to secrete a certain level of IL-2 and IFN- gamma.
In view of the above characteristics of CD11c~ (low) Ia~ (low) CD11b~ (high) DC, we speculate that the cell may have a negative immunomodulatory function. We add CD11c~ (low) Ia~ (low) CD11b~ (low) to the co culture system of 323-339 antigen specific peptide specificity / 323-339. The proliferation of T cells was inhibited and the secretion of intracellular cytokines in T cells was also detected. The results showed that CD11c~ (low) Ia~ (low) CD11b~ (high) DC still stimulated T cells to secrete a certain level of IL-2 and IFN- gamma. Y DC, DCreg).
In order to determine whether the regulatory DC induced by our in vitro co culture system has a corresponding DC subgroup in the tumor tumor, we analyze the CD11c~+ cell group in the tumor mononuclear cells based on the phenotypic characteristics of this regulatory DC. The most important characteristic of this regulatory DC is the high expression of CD11b, the low expression of CD11c and Ia, so we use it. CD11b-APC, CD11c-FITC and Ia-PE tricolor labeled CD11c~+ cell groups in tumor mononuclear cells. FACS analysis found a group of CD11b~ (high) CD11c~ (low) cells in this group. We further analyzed the expression of CD11b~ (high) CD11c~ cells. We found three groups of cells with low, medium and high continuous expression. Which group of cells have the similar functional characteristics with the regulatory DC induced by coculture system in vitro, we intend to use FACSVantage to separate these three groups of cells, analyze their cytokine secretion and inhibitory function, and finally confirm that there is a group of phenotypes and functions in the tumor microring. Similar tumor infiltrates into DC subgroup induced by co culture system of tumor cells in vitro. DCreg
The above studies have shown that the tumor microenvironment can indeed induce the differentiation of immature DC to DCreg, so in what form does the tumor cells induce the differentiation of immature DC to DCreg, the immunosuppressive soluble molecules secreted by the tumor cells, or the inhibitory membrane molecules expressed on the surface of the tumor cells, or both of them play a role together. First, we found that 3LL tumor cells express M-CSF, TGF- beta, VEGF and other cytokines with immunosuppressive effects by semi quantitative RT-PCR detection. In order to identify the form of 3LL tumor cells to induce the differentiation of immature DC to DCreg, we chose the Transwell system to carry out a study. Cell can block the release of soluble factors of tumor cells and can be used to study the role of tumor surface mask molecules. But because immature DC has a strong phagocytosis, the fixed tumor cells will be phagocytic by immature DC. Therefore, we abandoned this research pattern, using the Transwell system, and the Transwell system can make two kinds of cells effective. Isolation, avoiding direct contact between cells and cells, can be used to study the role of soluble factors secreted by tumor cells. We add immature DC directly into 24 foramen of cancer cells or add to the Transwell cells above the tumor cells and compare the inhibitory function of the two cells. The result is that the two groups of D are treated. C can effectively inhibit the proliferation of T cells, and there is no significant difference in the level of inhibition, indicating that the soluble factors secreted by the tumor cells are sufficient to inhibit the transformation of immature DC to DCreg. Which or which kinds of soluble factors are involved in the transformation of immature DC to DCreg, we will use neutralizing antibodies and / / Blocking and joint blocking experiments were performed or depressant.
The tumor microenvironment, which is secreted by the immunosuppressive soluble factors, can induce the immature DC into the tumor to convert into regulatory DC. This group of DC hypersecreting IL-10, PGE2 and NO can obviously inhibit the proliferation of antigen specific CD4~+T cells in vitro. We speculate whether this group DC not only does not have immune surveillance and immune defense. In order to investigate whether CD11c~ (low) Ia~ (low) CD11b~ (high) DC promotes tumor metastasis, we have established a lung metastasis model of 3LL tumor. We first injected the immature DC or DCreg tail vein to mice, and then injected the mouse 3LL lung cancer cells into the tail vein in 24 small hours, and then killed the mice after 20 days. The tumor metastasis of lung was observed with the method of ink infusion. The results showed that the metastasis of DCreg group was more than that of the immature DC group. It suggested that the group CD11c~ (low) Ia~ (low) CD11b~ (high) DC not only did not have immune surveillance and immune defense, but promoted the metastasis of tumor cells.
To sum up, the tumor cells can induce immature DC into the tumor through the composition and / or inducible secretion of some chemokines. Under the action of the soluble factors released by the tumor cells, the immature DC further differentiates into the CD11c~ (low) Ia~ (low) CD11b~ (high) regulatory IL-10, PGE2 and NO, but the low secretion of IL-12. It can inhibit the proliferation of antigenic peptide specific CD4~+T cells in vitro, and can promote tumor metastasis in vivo. This study also preliminarily demonstrated that there may be a corresponding regulatory DC subgroup in tumor bearing mice, which provides another new mechanism and explanation for tumor immune escape.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392;R730.3
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