cAMP在小剂量环磷酰胺逆转NHL动物模型免疫逃逸过程中的作用研究
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摘要
非霍奇金淋巴瘤(non-Hodgkin's lymphoma, NHL)是来源于B、T淋巴细胞的免疫系统恶性肿瘤,在NHL发生发展过程中,淋巴瘤细胞通过多种免疫机制以逃避机体的免疫监视系统。调节性T细胞(regulatory T cells, Tregs)在淋巴瘤的免疫逃逸和免疫耐受过程中发挥重要作用。许多研究致力于减少Tregs数量及/或功能,提高效应T细胞(effector T cells, Teff)的功能,从而阻断淋巴瘤免疫耐受形成的关键通路,期望对NHL免疫疗法起到关键性的辅助作用。Bopp T等的研究发现Tregs可以通过细胞间的缝隙连接将环磷酸腺苷(cyclic adenosine monophosphate, cAMP)转运至Teff细胞内,从而提高了Teff细胞内cAMP含量,抑制了白介素2的合成。提示免疫细胞内的cAMP含量与Tregs介导的免疫逃逸过程有着密切关系。
近年来研究证实,小剂量环磷酰胺(cyclophosphamide, CY)在肿瘤及自身免疫病的治疗中发挥免疫调节作用。CY是细胞毒类烷化剂,广泛应用于淋巴瘤化疗中,但是尚无小剂量环磷酰胺在淋巴瘤动物模型中免疫调节机制的相关研究。本研究中我们将小剂量CY应用于NHL动物模型中,探讨了CY对Tregs细胞的作用机制。本研究为创新性研究。
在淋巴系统恶性肿瘤发展的过程中涉及多种信号途径,细胞是存活还是凋亡取决于细胞内及细胞间多种信号途径之间的平衡网络。其中cAMP信号途径在淋巴瘤细胞凋亡过程中的作用日益受到重视,提高淋巴瘤细胞内的cAMP浓度有助于促进恶性淋巴细胞的凋亡,从而达到靶向治疗的目的。
本研究通过应用分子生物学、细胞生物学、动物实验以及免疫学技术,阐明了cAMP信号途径在小剂量环磷酰胺逆转NHL动物模型中免疫逃逸过程的作用。同时对小剂量环磷酰胺和cAMP拟似物的协同作用进行了初步探讨。寻求淋巴瘤免疫治疗的新方法,提高临床治疗效果。
第一部分：小剂量环磷酰胺逆转NHL动物模型免疫逃逸过程的作用研究
目的：本研究中,为了研究小剂量环磷酰胺(20mg/kg)对Tregs的抑制作用,我们选用小鼠B细胞淋巴瘤细胞株A20细胞建立侵袭性淋巴瘤小鼠模型。首先比较了治疗前后各组小鼠的荷瘤生存期及肿瘤直径变化。并进一步制备引流淋巴结的单细胞悬液,通过磁珠分选,获得纯化的Tregs和Teff细胞。检测免疫细胞的Foxp3及IL-2表达等指标,观察小剂量CY治疗前后Tregs和效应T细胞的功能变化。
材料与方法：
1.细胞培养和动物实验
2.单细胞制备
3.蛋白提取及蛋白印记分析
4.提取RNA,进行RT-PCR
5. Tregs及效应T细胞(Teff)的分离
6.流式细胞仪分析
7.统计学分析
结果：
1.A20细胞培养及小鼠模型的建立(图1)
(1)小鼠B系淋巴瘤细胞株A20由中山大学肿瘤研究所馈赠,应用含10%灭活的胎牛血清、青霉素(100U/m1)以及链霉素(100mg/mL)的RPMI-1640(Gibco)全培养基,在37℃,5%C02条件下培养。每2-3天传代一次。所有实验均选用对数生长期的细胞。
(2)制备A20细胞悬液：在A20细胞株处于对数期生长的情况下,使用不含血清的1640培养基洗净细胞,离心,170g,10min,调整密度至5×10'/mL。
(3)选取6-8周龄BALB/c近交系小鼠,在右背部皮下注射A20细胞悬液100μL。正常组以PBS溶液代替A20细胞悬液。肿瘤植入后7天,给予20mg/kg CY。
2. BALB/c小鼠NHL模型的观察
对接种A20细胞的小鼠的成瘤情况进行观察,成瘤率为90%。
皮下注射A20细胞悬液后7天、14天、21天、28天分别测量两组荷瘤小鼠的肿瘤直径,CY治疗后肿瘤直径明显缩小,生存期延长,延缓了肿瘤发展(图2A和B)。
3.流式细胞术检测
3.1荷瘤小鼠引流淋巴结内CD+CD25+T细胞比值变化
在荷瘤小鼠的引流淋巴结内,CD4+CD25+T细胞占淋巴细胞的比例肿瘤组明显高于正常对照组,CY治疗后下降,8天达到最低点,差异有统计学意义(p=0.0004)(图3)。
3.2MACS提取CD4+CD25-T细胞,CD4+CD25+T细胞的纯度测定
通过流式细胞术证实,使用MACS技术分离后得到的CD4+CD25-T细胞纯度在98%以上,CD4+CD25+T细胞纯度在90%以上。
4.实时定量RT-PCR检测Foxp3表达
Foxp3mRNA表达量在荷瘤小鼠引流淋巴结中表达高于对照组。CY治疗可以明显降低Foxp3表达,在治疗8天时达到抑制最低点,后Foxp3表达逐渐恢复至治疗前(图4)。
5. Western Blot技术检测各类T细胞内FoxP3蛋白的表达
MACS技术分离得到的淋巴结内CD4+CD25+T细胞,可见到FoxP3蛋白表达。经小剂量CY治疗后Foxp3蛋白表达量随着时间有变化。第8天最低,第14天恢复(图5)。
6.实时荧光定量PCR检测IL-2表达
BALB/c小鼠IL-2mRNA表达量显著高于荷瘤组。随着CY作用时间不同,IL-2表达量显示出先升高后降低的趋势,CY治疗第8天达到最高水平,与荷瘤小鼠比较有显著差异(图6)。
结论：
1.A20B细胞淋巴瘤小鼠模型是一种良好的NHL动物模型。
2. Tregs在淋巴瘤动物模型中高表达,与肿瘤免疫耐受相关。
3.小剂量环磷酰胺可短暂下调动物模型中Tregs比例及功能,刺激效应T细胞功能,有望逆转免疫耐受。
4.小剂量环磷酰胺延长动物模型的荷瘤生存期,是通过免疫调节发挥作用。
第二部分：cAMP在小剂量CY抑制Tregs过程中的作用研究和cAMP拟似物对NHL细胞凋亡作用的研究
目的：淋巴瘤小鼠模型中小剂量CY发挥了免疫调节作用,在本部分,我们进一步研究了免疫细胞内cAMP含量在CY作用过程中的变化,以明确CY的作用机制,为cAMP信号途径与环磷酰胺途径的协同作用奠定理论基础。肿瘤细胞是存活还是凋亡取决于细胞内及细胞间多种信号途径之间的平衡网络。其中cAMP信号途径在淋巴瘤细胞凋亡过程中的作用日益受到重视。本研究中,我们将临床常用的cAMP拟似物作用于A20淋巴瘤细胞株,并研究了CY和cAMP拟似物对动物模型的肿瘤组织中磷酸化及未磷酸化AKT的变化,探讨淋巴瘤细胞的凋亡机制,从而达到靶向治疗的目的。
材料与方法：
1.细胞培养及动物实验
2.免疫细胞内cAMP含量测定
3.肿瘤组织蛋白提取及蛋白印记分析
4.流式细胞仪检测A20细胞凋亡及周期阻滞
5.免疫组化研究
6.统计学分析
结果：
1.不同免疫细胞内cAMP含量变化
我们检测了小剂量CY治疗前后免疫细胞内的cAMP含量变化。明确cAMP信号途径在小剂量环磷酰胺作用过程中的重要作用。
1.1CD4+CD25-T细胞内cAMP含量变化：
BALB/c小鼠Teff细胞内较荷瘤小鼠低,CY治疗4天后细胞内cAMP含量逐渐降低,第8天降至最低,治疗14天时再次升高。CY治疗第8天与治疗14天比较,差异有统计学意义。
1.2CD4+CD25+T细胞内cAMP含量变化：
荷瘤小鼠较BALB/c小鼠Tregs细胞内含量高,CY治疗4天后细胞内cAMP含量逐渐降低至第8天最低,治疗14天时再次升高。CY8天与14天比较,差异有统计学意义(图8)。
2.A20细胞株凋亡
在不同浓度的cAMP拟似物诱导下,A20细胞出现不同程度的凋亡,周期阻滞不明显。
3.肿瘤组织中AKT磷酸化及未磷酸化形式的蛋白含量改变
小剂量CY对NHL动物模型中的肿瘤组织AKT形式磷酸化没有影响。而cAMP拟似物可以抑制磷酸化AKT的表达。
4.免疫组化结果
小剂量CY对NHL动物模型中的肿瘤组织AKT形式磷酸化没有影响。而cAMP拟似物可以抑制磷酸化AKT的表达。
结论：
1. cAMP拟似物诱导淋巴瘤细胞株凋亡
2.小剂量CY对NHL动物模型中的肿瘤组织AKT形式磷酸化没有影响。
3. cAMP拟似物可作为良好的候选药物。
4. cAMP信号途径在小剂量环磷酰胺逆转免疫耐受中发挥重要作用。
5.以似物和小剂量环磷酰胺的协同作用将在淋巴瘤治疗中有应用前景。
Non-Hodgkin's lymphoma (NHL) is a group of malignant tumors with immune disorders, deriving from B and T lymphocytes. The lymphoma cells mobilize many mechanisms to escape from the immune system. Now there is substantial evidence that the CD4+CD25+regulatory T cells (Tregs) play key roles in the control of tumor immune tolerance and immune evasion. Suppression of the number and/or function of Tregs may be critical for successful immunological therapy in lymphoma. Many researches focus on the removal of Tregs in animal models, resulting in improved function of CD4+effector cells (Teff), ultimately enhance the antitumor responses. Therefore, elucidating the mechanism involved in Treg-induced immune evasion in tumor progression, may have important implications in the design of effective immunotherapeutic approaches against cancer. Bopp T et al show that Tregs can transfer cyclic adenosine monophosphate (cAMP) to Teff cells through cell contact-dependent gap junction, followed by increase of intracellular concentration of cAMP in Teff cells, inhibiting the interleukin2(IL-2) synthesis in Teff cells. So the Treg cells may suppress the immune network through the intercellular transport of cAMP, indicating the correlation between intracellular cAMP concentration and immune-evasion role of Tregs.
Many researches focus on the depletion of Treg by anti-CD25monoclonal antibody (PC61) or low dose CY in tumor and some autoimmune conditions. Being an alkylating drug agent, CY is used in lymphoma chemotherapy. But there isn't investigation of low-dose CY in lymphoma animal models and patients. In our study, we chose A20B cell NHL lymphoma mouse as an aggressive tumor model, to investigate the depletion of Tregs by low-dose CY (20mg/kg). In the progress of lymphocytic malignancy several signal pathways are involved. Balance of the signaling pathways decides the fate of malignant cells, to survive or to undergo apoptosis. Recently, the role of the cyclic adenosine monophosphate (cAMP) signal pathway in the pathogenesis of lymphoma and apoptosis-inducing therapy has been researched. Increased intracellular cAMP levels induce cell-cycle arrest and apoptosis in several types of lymphoma cells. In our study, we established an aggressive transplantable murine lymphoma model with A20cells and investigated the effects of low-dose CY on tumor immunity. Treatment with20mg/kg CY inhibited tumor growth, increased the survival rates of A20tumor-bearing BALB/c mice, and ultimately increased the IL-2secretion of CD4+Teff cells. The IL-2increase was synchronized with the FOXP3decrease. With their potential roles in reversing tumor immune evasion and promoting apoptosis, and due to the potential clinical cooperation between the cAMP pathway and the CY-Tregs pathway, cAMP analogs/PDE4B inhibitors and low-dose CY may be good candidate agents for lymphoma therapy.
Part I:Investigation of low-dose
cyclophosphamide-reversed immune evasion in B cell non-Hodgkin's lymphoma mouse model
Objective:In our study, we chose A20B cell NHL lymphoma mouse as an aggressive tumor model, to investigate the depletion of Tregs by low-dose CY (20mg/kg). We observed the tumor diameters and survival rates of tumor-bearing mice. Then MACS isolation was done to obtain purified Tregs and/or Teffs. Foxp3and IL-2expression of immune cells was compared by Western Blot and RT-PCR between CY-treated and tumor-beared mice.
Material and methods:
1. Cell culture and animal experiments
2. Single cell preparation
3. Proin extraction and Western Blot
4. RNA extraction and RT-PCR
5. Treg cells and Teff cells isolation
6. Flow cytometric analysis
7. Statistical Analysis
Results:
1. A20cells culture and lymphoma mouse model The mouse B-cell lymphoma cell line, A20, was a generous gift from Zhongshan University (Guangzhou, China). This tumor cell line is derived from a spontaneous reticulum cell neoplasm in BALB/c mice[1].The A20cells were cultured in RPMI1640medium (Hyclone, Logan, UT) that was supplemented with10%heat-inactivated fetal bovine serum,100U/ml penicillin,100μg/ml streptomycin and2mM L-glutamine at37℃in a5%CO2-humidified incubator. A20lymphoma cells were implanted into BALB/c mice at concentrations of5×106cells per mouse in100μl of RPMI1640by subcutaneous injection into the left flanks. After7days, the tumor-bearing mice received20mg/kg of CY or NS via the intraperitoneal (i.p.) route each week.
2. The tumor growth of BALB/c mice. We measured the tumor diameters and calculated the survival proportion as shown in Fig2. The treatment of20mg/kg CY caused slight delay on the growth of tumors, ultimately prolonged survival proportion of A20-bearing BALB/c mice.
3. FACS analysis. Lymphocytes of A20tumor-bearing or control mice with and without CY treatment (20mg/kg) were analyzed by flow cytometry for the presence of Tregs. The subcutaneous A20tumors increased the percentage of Tregs in the draining lymph nodes,4days after CY injection, the percentage of Tregs decreased and remained low on day8, then increased on day14.
After the MACS isolation, we performed FACS to validate the purity of isolated Tregs and Teff cells. The purity of Teffs was higher than98%, and the purity of Tregs was higher than90%.
4. Foxp3expression was transiently affected by low-dose CY. To confirm the nature of Tregs alteration, we performed Western Blot and Real-time PCR for the analysis of the Treg-specific marker Foxp3. We detected (3-actin content to assess cell equivalency among the samples, then detected Treg-specific marker Foxp3mRNA or protein expression using specific primers or mAb. Treatment with CY was found markedly to decrease the Foxp3expression from day4, reduced the mRNA and protein expression of Foxp3to a minimum level on day8, then recovered or exceeded to pretreatment levels in lymphoid organs by day14.
5. IL-2expression was affected by low-dose CY.
Along with the decrease of Tregs, we found the increase of IL-2expression of T effector cells. Tumor-challenge decreased the secretion of IL-2of CD4+effecter T cells (p<0.0001), but CY-treatment developed the function of effecter T cells (Fig.4). The elevation peaked on day8(p<0.0001), then decreased gradually, disappeared after14days. Then the transient interval may provide chance for effective anti-immune response which needs Teff cells activation.
Conclusions:
1. A20B cell NHL lymphoma mouse model was a good NHL animal model.
2. A20lymphoma model had increased Tregs which induced the immue-evasion.
3. Low-dose CY could transiently decrease the function and number of the Tregs and increase the expression of IL-2, ultimately reversed the immune-evasion mediated by Tregs in B cell lymphoma mouse model.
4. Loe-dose CY could prolong the survival of mouse model, proved the immune-enhancing function of low-dose CY in the treatment of lymphoma mouse model.
Part Ⅱ:Involvement of cAMP in the low-dose
cyclophosphamide-reversed immune evasion and role of cAMP analogs in apoptosis of NHL cells
Objective:Loe-dose CY could prolong the survival of mouse model, proved the immune-enhancing function of low-dose C Y in the treatment of lymphoma mouse model. We further investigated intracellular cAMP concentration of immune cells with or without CY treatment, in order to provide theoretical foundation for the cooperation of cAMP analogs and low-dose CY. Balance of the signaling pathways decides the fate of malignant cells, to survive or to undergo apoptosis. Recently, the role of the cyclic adenosine monophosphate (cAMP) signal pathway in the pathogenesis of lymphoma and apoptosis-inducing therapy has been researched. In our study, we injected the cAMP analogs into A20NHL mice model, then detected the change of AKT, in order to investigate the apoptosis mechanism of lymphoma. cAMP analogs/PDE4B inhibitors and low-dose CY may be good candidate agents for lymphoma therapy.
Material and methods:
1. Cell culture and animal experiments
2. Intracellular cAMP concentration assay
3. Proin extraction and Western Blot
4. Flow cytometric analysis
5. Immunohistochemistry
6. Statistical Analysis
Results:
1. The change of intracellular cAMP concentration of immune cells. In this study, we measured intracellular cAMP concentration of immune cells with or without CY treatment. Consistent with the change of Foxp3and IL-2after CY treatment, the intracellular cAMP concentration altered regularly after CY-treatment. We found that tumor-challenge increased the cAMP concentration of immune cells. CY-treatment decreased the cAMP concentration from day4, and T effecter cells harbored the lowest cAMP concentration on day8. The down-regulation was temporary, faded on day14. Then20mg/kg CY created a transient window for optimal T effecter's differentiation and postponing the tumor growth.
2. Apoptosis of A20cell line The mouse B-cell lymphoma cell line, A20, showed increased apoptosis rate, under role of cAMP analogs
3. The change of Akt phosphorylation protein wasn't affectecd by low-dose CY.
4. The change of Akt phosphorylation protein was investigated by immunohistochemistry
Conclusions:
1. cAMP analogs induce A20B cell line to apoptosis.
2. The change of Akt phosphorylation protein wasn't affectecd by low-dose CY.
3. cAMP analogues are good candidate agents for lymphoma therapy.
4. cAMP is involved in low-dose CY-reversed immue evasion.
5. Cooperation of cAMP analogs and low-dose CY maybe protective candidate for the immune-therapy of B cell lymphoma.
近年来研究证实,小剂量环磷酰胺(cyclophosphamide, CY)在肿瘤及自身免疫病的治疗中发挥免疫调节作用。CY是细胞毒类烷化剂,广泛应用于淋巴瘤化疗中,但是尚无小剂量环磷酰胺在淋巴瘤动物模型中免疫调节机制的相关研究。本研究中我们将小剂量CY应用于NHL动物模型中,探讨了CY对Tregs细胞的作用机制。本研究为创新性研究。
在淋巴系统恶性肿瘤发展的过程中涉及多种信号途径,细胞是存活还是凋亡取决于细胞内及细胞间多种信号途径之间的平衡网络。其中cAMP信号途径在淋巴瘤细胞凋亡过程中的作用日益受到重视,提高淋巴瘤细胞内的cAMP浓度有助于促进恶性淋巴细胞的凋亡,从而达到靶向治疗的目的。
本研究通过应用分子生物学、细胞生物学、动物实验以及免疫学技术,阐明了cAMP信号途径在小剂量环磷酰胺逆转NHL动物模型中免疫逃逸过程的作用。同时对小剂量环磷酰胺和cAMP拟似物的协同作用进行了初步探讨。寻求淋巴瘤免疫治疗的新方法,提高临床治疗效果。
第一部分：小剂量环磷酰胺逆转NHL动物模型免疫逃逸过程的作用研究
目的：本研究中,为了研究小剂量环磷酰胺(20mg/kg)对Tregs的抑制作用,我们选用小鼠B细胞淋巴瘤细胞株A20细胞建立侵袭性淋巴瘤小鼠模型。首先比较了治疗前后各组小鼠的荷瘤生存期及肿瘤直径变化。并进一步制备引流淋巴结的单细胞悬液,通过磁珠分选,获得纯化的Tregs和Teff细胞。检测免疫细胞的Foxp3及IL-2表达等指标,观察小剂量CY治疗前后Tregs和效应T细胞的功能变化。
材料与方法：
1.细胞培养和动物实验
2.单细胞制备
3.蛋白提取及蛋白印记分析
4.提取RNA,进行RT-PCR
5. Tregs及效应T细胞(Teff)的分离
6.流式细胞仪分析
7.统计学分析
结果：
1.A20细胞培养及小鼠模型的建立(图1)
(1)小鼠B系淋巴瘤细胞株A20由中山大学肿瘤研究所馈赠,应用含10%灭活的胎牛血清、青霉素(100U/m1)以及链霉素(100mg/mL)的RPMI-1640(Gibco)全培养基,在37℃,5%C02条件下培养。每2-3天传代一次。所有实验均选用对数生长期的细胞。
(2)制备A20细胞悬液：在A20细胞株处于对数期生长的情况下,使用不含血清的1640培养基洗净细胞,离心,170g,10min,调整密度至5×10'/mL。
(3)选取6-8周龄BALB/c近交系小鼠,在右背部皮下注射A20细胞悬液100μL。正常组以PBS溶液代替A20细胞悬液。肿瘤植入后7天,给予20mg/kg CY。
2. BALB/c小鼠NHL模型的观察
对接种A20细胞的小鼠的成瘤情况进行观察,成瘤率为90%。
皮下注射A20细胞悬液后7天、14天、21天、28天分别测量两组荷瘤小鼠的肿瘤直径,CY治疗后肿瘤直径明显缩小,生存期延长,延缓了肿瘤发展(图2A和B)。
3.流式细胞术检测
3.1荷瘤小鼠引流淋巴结内CD+CD25+T细胞比值变化
在荷瘤小鼠的引流淋巴结内,CD4+CD25+T细胞占淋巴细胞的比例肿瘤组明显高于正常对照组,CY治疗后下降,8天达到最低点,差异有统计学意义(p=0.0004)(图3)。
3.2MACS提取CD4+CD25-T细胞,CD4+CD25+T细胞的纯度测定
通过流式细胞术证实,使用MACS技术分离后得到的CD4+CD25-T细胞纯度在98%以上,CD4+CD25+T细胞纯度在90%以上。
4.实时定量RT-PCR检测Foxp3表达
Foxp3mRNA表达量在荷瘤小鼠引流淋巴结中表达高于对照组。CY治疗可以明显降低Foxp3表达,在治疗8天时达到抑制最低点,后Foxp3表达逐渐恢复至治疗前(图4)。
5. Western Blot技术检测各类T细胞内FoxP3蛋白的表达
MACS技术分离得到的淋巴结内CD4+CD25+T细胞,可见到FoxP3蛋白表达。经小剂量CY治疗后Foxp3蛋白表达量随着时间有变化。第8天最低,第14天恢复(图5)。
6.实时荧光定量PCR检测IL-2表达
BALB/c小鼠IL-2mRNA表达量显著高于荷瘤组。随着CY作用时间不同,IL-2表达量显示出先升高后降低的趋势,CY治疗第8天达到最高水平,与荷瘤小鼠比较有显著差异(图6)。
结论：
1.A20B细胞淋巴瘤小鼠模型是一种良好的NHL动物模型。
2. Tregs在淋巴瘤动物模型中高表达,与肿瘤免疫耐受相关。
3.小剂量环磷酰胺可短暂下调动物模型中Tregs比例及功能,刺激效应T细胞功能,有望逆转免疫耐受。
4.小剂量环磷酰胺延长动物模型的荷瘤生存期,是通过免疫调节发挥作用。
第二部分：cAMP在小剂量CY抑制Tregs过程中的作用研究和cAMP拟似物对NHL细胞凋亡作用的研究
目的：淋巴瘤小鼠模型中小剂量CY发挥了免疫调节作用,在本部分,我们进一步研究了免疫细胞内cAMP含量在CY作用过程中的变化,以明确CY的作用机制,为cAMP信号途径与环磷酰胺途径的协同作用奠定理论基础。肿瘤细胞是存活还是凋亡取决于细胞内及细胞间多种信号途径之间的平衡网络。其中cAMP信号途径在淋巴瘤细胞凋亡过程中的作用日益受到重视。本研究中,我们将临床常用的cAMP拟似物作用于A20淋巴瘤细胞株,并研究了CY和cAMP拟似物对动物模型的肿瘤组织中磷酸化及未磷酸化AKT的变化,探讨淋巴瘤细胞的凋亡机制,从而达到靶向治疗的目的。
材料与方法：
1.细胞培养及动物实验
2.免疫细胞内cAMP含量测定
3.肿瘤组织蛋白提取及蛋白印记分析
4.流式细胞仪检测A20细胞凋亡及周期阻滞
5.免疫组化研究
6.统计学分析
结果：
1.不同免疫细胞内cAMP含量变化
我们检测了小剂量CY治疗前后免疫细胞内的cAMP含量变化。明确cAMP信号途径在小剂量环磷酰胺作用过程中的重要作用。
1.1CD4+CD25-T细胞内cAMP含量变化：
BALB/c小鼠Teff细胞内较荷瘤小鼠低,CY治疗4天后细胞内cAMP含量逐渐降低,第8天降至最低,治疗14天时再次升高。CY治疗第8天与治疗14天比较,差异有统计学意义。
1.2CD4+CD25+T细胞内cAMP含量变化：
荷瘤小鼠较BALB/c小鼠Tregs细胞内含量高,CY治疗4天后细胞内cAMP含量逐渐降低至第8天最低,治疗14天时再次升高。CY8天与14天比较,差异有统计学意义(图8)。
2.A20细胞株凋亡
在不同浓度的cAMP拟似物诱导下,A20细胞出现不同程度的凋亡,周期阻滞不明显。
3.肿瘤组织中AKT磷酸化及未磷酸化形式的蛋白含量改变
小剂量CY对NHL动物模型中的肿瘤组织AKT形式磷酸化没有影响。而cAMP拟似物可以抑制磷酸化AKT的表达。
4.免疫组化结果
小剂量CY对NHL动物模型中的肿瘤组织AKT形式磷酸化没有影响。而cAMP拟似物可以抑制磷酸化AKT的表达。
结论：
1. cAMP拟似物诱导淋巴瘤细胞株凋亡
2.小剂量CY对NHL动物模型中的肿瘤组织AKT形式磷酸化没有影响。
3. cAMP拟似物可作为良好的候选药物。
4. cAMP信号途径在小剂量环磷酰胺逆转免疫耐受中发挥重要作用。
5.以似物和小剂量环磷酰胺的协同作用将在淋巴瘤治疗中有应用前景。
Non-Hodgkin's lymphoma (NHL) is a group of malignant tumors with immune disorders, deriving from B and T lymphocytes. The lymphoma cells mobilize many mechanisms to escape from the immune system. Now there is substantial evidence that the CD4+CD25+regulatory T cells (Tregs) play key roles in the control of tumor immune tolerance and immune evasion. Suppression of the number and/or function of Tregs may be critical for successful immunological therapy in lymphoma. Many researches focus on the removal of Tregs in animal models, resulting in improved function of CD4+effector cells (Teff), ultimately enhance the antitumor responses. Therefore, elucidating the mechanism involved in Treg-induced immune evasion in tumor progression, may have important implications in the design of effective immunotherapeutic approaches against cancer. Bopp T et al show that Tregs can transfer cyclic adenosine monophosphate (cAMP) to Teff cells through cell contact-dependent gap junction, followed by increase of intracellular concentration of cAMP in Teff cells, inhibiting the interleukin2(IL-2) synthesis in Teff cells. So the Treg cells may suppress the immune network through the intercellular transport of cAMP, indicating the correlation between intracellular cAMP concentration and immune-evasion role of Tregs.
Many researches focus on the depletion of Treg by anti-CD25monoclonal antibody (PC61) or low dose CY in tumor and some autoimmune conditions. Being an alkylating drug agent, CY is used in lymphoma chemotherapy. But there isn't investigation of low-dose CY in lymphoma animal models and patients. In our study, we chose A20B cell NHL lymphoma mouse as an aggressive tumor model, to investigate the depletion of Tregs by low-dose CY (20mg/kg). In the progress of lymphocytic malignancy several signal pathways are involved. Balance of the signaling pathways decides the fate of malignant cells, to survive or to undergo apoptosis. Recently, the role of the cyclic adenosine monophosphate (cAMP) signal pathway in the pathogenesis of lymphoma and apoptosis-inducing therapy has been researched. Increased intracellular cAMP levels induce cell-cycle arrest and apoptosis in several types of lymphoma cells. In our study, we established an aggressive transplantable murine lymphoma model with A20cells and investigated the effects of low-dose CY on tumor immunity. Treatment with20mg/kg CY inhibited tumor growth, increased the survival rates of A20tumor-bearing BALB/c mice, and ultimately increased the IL-2secretion of CD4+Teff cells. The IL-2increase was synchronized with the FOXP3decrease. With their potential roles in reversing tumor immune evasion and promoting apoptosis, and due to the potential clinical cooperation between the cAMP pathway and the CY-Tregs pathway, cAMP analogs/PDE4B inhibitors and low-dose CY may be good candidate agents for lymphoma therapy.
Part I:Investigation of low-dose
cyclophosphamide-reversed immune evasion in B cell non-Hodgkin's lymphoma mouse model
Objective:In our study, we chose A20B cell NHL lymphoma mouse as an aggressive tumor model, to investigate the depletion of Tregs by low-dose CY (20mg/kg). We observed the tumor diameters and survival rates of tumor-bearing mice. Then MACS isolation was done to obtain purified Tregs and/or Teffs. Foxp3and IL-2expression of immune cells was compared by Western Blot and RT-PCR between CY-treated and tumor-beared mice.
Material and methods:
1. Cell culture and animal experiments
2. Single cell preparation
3. Proin extraction and Western Blot
4. RNA extraction and RT-PCR
5. Treg cells and Teff cells isolation
6. Flow cytometric analysis
7. Statistical Analysis
Results:
1. A20cells culture and lymphoma mouse model The mouse B-cell lymphoma cell line, A20, was a generous gift from Zhongshan University (Guangzhou, China). This tumor cell line is derived from a spontaneous reticulum cell neoplasm in BALB/c mice[1].The A20cells were cultured in RPMI1640medium (Hyclone, Logan, UT) that was supplemented with10%heat-inactivated fetal bovine serum,100U/ml penicillin,100μg/ml streptomycin and2mM L-glutamine at37℃in a5%CO2-humidified incubator. A20lymphoma cells were implanted into BALB/c mice at concentrations of5×106cells per mouse in100μl of RPMI1640by subcutaneous injection into the left flanks. After7days, the tumor-bearing mice received20mg/kg of CY or NS via the intraperitoneal (i.p.) route each week.
2. The tumor growth of BALB/c mice. We measured the tumor diameters and calculated the survival proportion as shown in Fig2. The treatment of20mg/kg CY caused slight delay on the growth of tumors, ultimately prolonged survival proportion of A20-bearing BALB/c mice.
3. FACS analysis. Lymphocytes of A20tumor-bearing or control mice with and without CY treatment (20mg/kg) were analyzed by flow cytometry for the presence of Tregs. The subcutaneous A20tumors increased the percentage of Tregs in the draining lymph nodes,4days after CY injection, the percentage of Tregs decreased and remained low on day8, then increased on day14.
After the MACS isolation, we performed FACS to validate the purity of isolated Tregs and Teff cells. The purity of Teffs was higher than98%, and the purity of Tregs was higher than90%.
4. Foxp3expression was transiently affected by low-dose CY. To confirm the nature of Tregs alteration, we performed Western Blot and Real-time PCR for the analysis of the Treg-specific marker Foxp3. We detected (3-actin content to assess cell equivalency among the samples, then detected Treg-specific marker Foxp3mRNA or protein expression using specific primers or mAb. Treatment with CY was found markedly to decrease the Foxp3expression from day4, reduced the mRNA and protein expression of Foxp3to a minimum level on day8, then recovered or exceeded to pretreatment levels in lymphoid organs by day14.
5. IL-2expression was affected by low-dose CY.
Along with the decrease of Tregs, we found the increase of IL-2expression of T effector cells. Tumor-challenge decreased the secretion of IL-2of CD4+effecter T cells (p<0.0001), but CY-treatment developed the function of effecter T cells (Fig.4). The elevation peaked on day8(p<0.0001), then decreased gradually, disappeared after14days. Then the transient interval may provide chance for effective anti-immune response which needs Teff cells activation.
Conclusions:
1. A20B cell NHL lymphoma mouse model was a good NHL animal model.
2. A20lymphoma model had increased Tregs which induced the immue-evasion.
3. Low-dose CY could transiently decrease the function and number of the Tregs and increase the expression of IL-2, ultimately reversed the immune-evasion mediated by Tregs in B cell lymphoma mouse model.
4. Loe-dose CY could prolong the survival of mouse model, proved the immune-enhancing function of low-dose CY in the treatment of lymphoma mouse model.
Part Ⅱ:Involvement of cAMP in the low-dose
cyclophosphamide-reversed immune evasion and role of cAMP analogs in apoptosis of NHL cells
Objective:Loe-dose CY could prolong the survival of mouse model, proved the immune-enhancing function of low-dose C Y in the treatment of lymphoma mouse model. We further investigated intracellular cAMP concentration of immune cells with or without CY treatment, in order to provide theoretical foundation for the cooperation of cAMP analogs and low-dose CY. Balance of the signaling pathways decides the fate of malignant cells, to survive or to undergo apoptosis. Recently, the role of the cyclic adenosine monophosphate (cAMP) signal pathway in the pathogenesis of lymphoma and apoptosis-inducing therapy has been researched. In our study, we injected the cAMP analogs into A20NHL mice model, then detected the change of AKT, in order to investigate the apoptosis mechanism of lymphoma. cAMP analogs/PDE4B inhibitors and low-dose CY may be good candidate agents for lymphoma therapy.
Material and methods:
1. Cell culture and animal experiments
2. Intracellular cAMP concentration assay
3. Proin extraction and Western Blot
4. Flow cytometric analysis
5. Immunohistochemistry
6. Statistical Analysis
Results:
1. The change of intracellular cAMP concentration of immune cells. In this study, we measured intracellular cAMP concentration of immune cells with or without CY treatment. Consistent with the change of Foxp3and IL-2after CY treatment, the intracellular cAMP concentration altered regularly after CY-treatment. We found that tumor-challenge increased the cAMP concentration of immune cells. CY-treatment decreased the cAMP concentration from day4, and T effecter cells harbored the lowest cAMP concentration on day8. The down-regulation was temporary, faded on day14. Then20mg/kg CY created a transient window for optimal T effecter's differentiation and postponing the tumor growth.
2. Apoptosis of A20cell line The mouse B-cell lymphoma cell line, A20, showed increased apoptosis rate, under role of cAMP analogs
3. The change of Akt phosphorylation protein wasn't affectecd by low-dose CY.
4. The change of Akt phosphorylation protein was investigated by immunohistochemistry
Conclusions:
1. cAMP analogs induce A20B cell line to apoptosis.
2. The change of Akt phosphorylation protein wasn't affectecd by low-dose CY.
3. cAMP analogues are good candidate agents for lymphoma therapy.
4. cAMP is involved in low-dose CY-reversed immue evasion.
5. Cooperation of cAMP analogs and low-dose CY maybe protective candidate for the immune-therapy of B cell lymphoma.
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