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摘要:目的 在体外诱导形成三级淋巴器官（tertiary lymphoid organs, TLO），并评价其在抗肿瘤免疫中的功能。方法 利用慢病毒系统在NIH3T3细胞上过表达淋巴毒素-β受体（lymphotoxin-beta receptor, LTβR），并检测LTβR-NIH3T3细胞中LTβR的过表达效率；通过免疫印迹实验和qPCR探究过表达LTβR的NIH3T3细胞内非经典核因子（nuclear factor, NF）-κB信号通路的情况。构建B16-OVA黑色素瘤小鼠荷瘤模型，探究LTβR-NIH3T3细胞诱导TLO形成的能力及抗肿瘤效果。结果 利用慢病毒感染在NIH3T3细胞中过表达LTβR，流式检测发现GFP+细胞比例达99%。过表达LTβR能在NIH3T3细胞内激活非经典NF-κB信号通路。小鼠肿瘤模型结果表明，注射LTβR-NIH3T3细胞能在肿瘤附近诱导形成淋巴样组织，并促进了T细胞和MHCⅡ+巨噬细胞的浸润，明显抑制荷瘤小鼠的肿瘤生长，并延长小鼠生存期。结论 LTβR-NIH3T3细胞通过诱导TLO的形成，促进抗肿瘤免疫，为肿瘤免疫治疗提供新的思路。
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关键词:
- 三级淋巴器官 /
- 抗肿瘤免疫 /
- 非经典NF-κB信号通路
Abstract:Objective To induce the development of tertiary lymphoid organs (TLO) in a mouse model of melanoma and to evaluate TLO’s functions in antitumor immunity.Methods Lymphotoxin-beta receptor (LTβR) was overexpressed in NIH3T3 cells through the lentivirus system and the overexpression efficiency of LTβR in LTβR-NIH3T3 cells was examined. Western blot and qPCR were used to examine the non-canonical nuclear factor (NF)-κB signaling pathway in NIH3T3 cells overexpressing LTβR. B16-OVA melanoma mouse model was constructed to explore the induction of TLO and anti-tumor functions of TLO in LTβR-NIH3T3 cells.Results LTβR was overexpressed in NIH3T3 cells through the lentivirus system, and flow cytometry showed that the proportion of GFP+ cells reached 99%. The overexpression of LTβR activated the non-canonical NF-κB signaling pathway in NIH3T3 cells. Findings from the mouse tumor model suggest that the injection of LTβR-NIH3T3 cells successfully induced the development of lymphoid tissue around the tumor and enhanced the tumor infiltration of T cells and MHCⅡ+ macrophages, significantly inhibiting tumor growth and prolonging the survival of tumor-bearing mice.Conclusion LTβR-NIH3T3 cells promoted anti-tumor immunity by inducing TLO development, which may provide new perspectives for tumor immunotherapy. -
癌症是严重威胁人类健康的世界公共卫生问题之一。二级淋巴器官（secondary lymphoid organs, SLO）的发育起始于胚胎阶段，在出生后早期完成。临床上，在肿瘤基质、浸润边缘和/或核心中发现淋巴组织的增生，被称为三级淋巴器官（tertiary lymphoid organs, TLO）[1-2]。TLO是成体中出现的异位淋巴器官，通常发生于感染、器官移植、自身免疫性疾病和肿瘤等慢性炎症条件下，其细胞组成和功能与SLO相似[3-4]。 在发育机制上，非经典核因子（nuclear factor, NF）-κB 信号通路是SLO与TLO形成所必需的，该通路的激活依赖肿瘤坏死因子（tumor-necrosis factor, TNF）超家族成员，如淋巴毒素-β受体（lymphotoxin-beta receptor, LTβR）、淋巴毒素（lymphotoxin, LT）α1β2、LIGHT等[5-6]。基质细胞分泌趋化因子CXCL13，将淋巴组织诱导细胞（lymphoid tissue inducer, LTi）募集到炎症部位，LTi分泌LTα1β2，与基质细胞表面表达的LTβR相互作用，激活基质细胞产生血管内皮生长因子，促进高内皮小静脉（high endothelial venules, HEVs）的发育，并上调黏附分子VCAM-1、ICAM-1 和 MAdCAM-1[7-8]。LTβ-LTβR和IL-17-IL17R联合作用，活化各种趋化因子和趋化因子配体的表达，如CXCL13（结合CXCR5），CCL19和CCL21（结合CCR7）[9-10]。这些趋化因子从附近的HEVs募集更多的淋巴细胞，逐渐形成有序的淋巴组织[11]。TLO具有与适应性免疫反应的产生相关的特征，包括T细胞区、B细胞区和HEVs[12]。
在肺癌、结直肠癌、黑色素瘤和乳腺癌等多个不同肿瘤类型中，均鉴定到TLO，多数情况下TLO的存在与良好的预后相关，提示TLO可能具有诱导持久的抗肿瘤反应的能力[13-16]。目前已在多个课题组开发利用趋化因子、细胞因子、抗体和抗原提呈细胞等诱导形成TLO，从而促进淋巴细胞浸润，增强抗肿瘤免疫反应[17-21]。本研究通过在基质细胞中过表达LTβR诱导形成TLO，研究其形成的分子机制并评价其抗肿瘤免疫效果。今后可结合免疫检查点抑制剂，成为癌症治疗的有效途径。
1. 材料和方法
1.1 实验质粒、细胞和实验动物
实验所用质粒pGFP-Myc空白质粒、CopGFP-Myc-mLtbr质粒、PsPAX2质粒和VSVg质粒均为本实验室构建和保存。
实验所用小鼠胚胎成纤维细胞系NIH3T3和人胚肾细胞系HEK293T来源于中国典型培养物保藏中心（CCTCC）。小鼠黑色素瘤B16-OVA细胞由西南医院刘新东教授惠赠。
实验所用小鼠为野生型C57B6小鼠，购自恩斯维尔生物科技有限公司。小鼠的饲养、管理和所有动物实验都按四川大学动物伦理管理委员会要求进行。
1.2 实验试剂
免疫印迹实验抗体p100/p52（美国Cell signaling Technology公司）；LaminB（美国Santa cruz公司）；β-actin（美国SIGM公司）; 羊抗鼠IgG-HRP和羊抗兔IgG-HRP（北京索莱宝公司）；流式抗体FITC-CD11c、PE-CD11b、PerCP-Cy5.5-Ly6C、PE-Cy7-Ly6G、APC-Cy7-CD45、Brilliant Violet 510-I-A/I-E、Pacific Blue-F4/80、PE-Cy7-CD19、FITC-CD3、APC-CD4、Pacific Blue-CD8和Brilliant Violet 711-CD4（美国Biolegend公司）；PE-Cy7-Foxp3（美国ebioscience公司）；DMEM高糖培养基和RPMI-1640培养基（美国HyClone公司）；青霉素/链霉素（Corning）。
1.3 主要仪器
PCR仪（美国Eppendorf公司）；荧光定量PCR仪、蛋白电泳仪、蛋白质电泳系统和酶标仪（美国Bio-Rad公司）；流式细胞分析仪（美国BD公司）；电子分析天平（瑞士Mettler Toledo公司）；水浴超声仪（昆山超声仪器厂）；恒温水浴箱（美国PolyScience公司）；超纯水系统（美国Millipore公司）；恒温摇床（ZHCHENG）；小型高速离心机（德国Sorvall PICO公司）；小型低温高速离心机（德国Sorvall FRESCO公司）；低速离心机（德国Heraeus Megafuge公司）。
1.4 实验方法
1.4.1 NIH3T3细胞过表达LTβR及验证
将CopGFP-Myc-mLtbr质粒、PsPAX2质粒和VSVg质粒通过磷酸钙法转染HEK293T细胞，转染后8～12 h换新鲜含1%青霉素/链霉素和10%胎牛血清的高糖DMEM完全培养基，培养36 h后收集病毒上清。提前1 d将NIH3T3细胞铺于6孔板中，铺板细胞数为每孔1×106。吸走6孔板中的培养基，加入1 mL病毒上清，2 000×g，30 ℃离心60 min。离心结束，放入37 ℃细胞孵箱。6 h后换液，继续培养40 h后，消化感染后的NIH3T3细胞，进行流式分析检测感染效率（用BD FACSAria Ⅲ分选高表达GFP的细胞）。为进一步得到LTβR-NIH3T3稳定表达细胞系，利用流式分选出高表达LTβR的LTβR-NIH3T3细胞，通过qPCR进一步鉴定流式分选前后Ltbr的表达水平。以转染pGFP-Myc空白质粒的NIH3T3细胞（Vector-NIH3T3）作为对照。
1.4.2 Western blot检测LTβR-NIH3T3细胞非经典NF-κB通路
将未转染的NIH3T3细胞、Vector-NIH3T3细胞和LTβR-NIH3T3细胞裂解后，提取胞浆蛋白和核蛋白，BCA法测定蛋白浓度。免疫印迹实验检测细胞质P100加工以及P52/RelB入核情况。聚丙烯酰胺凝胶电泳分离蛋白，转膜后牛奶封闭1 h，4 ℃孵育一抗α-P100、α-P52、α-RelB、LaminB和β-actin过夜，洗膜，室温孵育二抗1 h，曝光。以目的蛋白与内参蛋白光密度值的比值作为目的蛋白的相对表达量。
1.4.3 qPCR检测LTβR-NIH3T3细胞非经典NF-κB通路
提前1 d将Vector-NIH3T3和LTβR-NIH3T3细胞铺于6孔板中，细胞数为每孔1×106。分别设不处理（Control）组和αnti-LTβR激活型抗体刺激24 h组，收集细胞Trizol裂解，酚氯仿提取RNA逆转录为cDNA，qPCR检测基因Ccl19、Ccl21、Madcam1、Icam1、Vcam1的mRNA表达水平，18S为内参基因。基因引物序列见表1。反应条件为：预变性95 ℃ 5 min；变性95 ℃ 15 s，退火59 ℃ 30 s，延伸72 ℃ 30 s；循环35次。结果用2−ΔΔCt法计算mRNA的相对表达量。
表 1 qPCR引物序列及长度Table 1. Sequences and length of qPCR primersGene Forward (5′→3′) Reverse (5′→3′) Length/bp Ltbr CATGCTAGCATGCGCCTGCCCCGGGCCTC TGAGCGGCCGCTCAGAGGTCTTGGCATCCTAGTG 212 Ccl19 GAAAGCCTTCCGCTACCTTC GAGGTGCACAGAGCTGATAG 92 Ccl21 TCCGAGGCTATAGGAAGCAA CTTCCTCAGGGTTTGCACAT 108 Madcam1 GAGCAAGAAGAGGAGATACAAGAG TGGTGACCTGGCAGTGAAG 117 Icam1 GTGCTTTGAGAACTGTGGCA GGTCCTTGCCTACTTGCTG 119 Vcam1 GGAAGCTGGAACGAAGTATCC AAACACTTGACCGTGACCG 109 18S ACAGGGAGAAAGCGCAAAAC TGTGGCCTTGTGGTGAAGAG 237 1.4.4 小鼠黑色素瘤模型建立和检测
SPF级的C57B6小鼠养至6～8周，打耳钉标记并剃毛。胰酶消化B16-OVA细胞，用PBS重悬至浓度为1×107 mL−1，吹匀后用胰岛素针吸取100 μL细胞悬液注射至小鼠裸露皮下。接瘤后第9天，将荷瘤小鼠随机分为两组：Vector-NIH3T3组（n=11），LTβR-NIH3T3组（n=15）。分别消化Vector-NIH3T3和LTβR-NIH3T3细胞，同样重悬至浓度为1×107 mL−1，在小鼠大腿后侧皮肤进行皮下注射。接瘤后第10天开始，每两天记录小鼠肿瘤体积及存活情况，小鼠肿瘤体积计算方法为V=0.5×a×b2（a为最长径，b为最短径），并绘制肿瘤生长曲线。接瘤后第21天，处死荷瘤小鼠，剖离小鼠肿瘤并测量肿瘤质量，分别取Vector-NIH3T3（Vec）组和LTβR-NIH3T3（LTβR）组的部分肿瘤加入体积分数为4%多聚甲醛固定用于病理切片和HE染色，其余肿瘤剪碎，加入终质量浓度为1 mg/mL的胶原酶Ⅳ和100 μg/mL的DNaseⅠ消化肿瘤，37 ℃摇晃孵育30 min，进行流式染色，分析肿瘤浸润淋巴细胞。同时绘制小鼠生存曲线，此为独立实验，从小鼠接瘤后第10天开始记录小鼠存活情况直至其死亡，但若肿瘤体积超过2 000 mm3，按动物伦理要求小鼠将被处死，在生存曲线上记录为死亡。
1.5 统计学方法
两个样本间比较采用双侧未配对t检验；多个样本间比较采用单因素方差分析（One-way ANOVA）；多组间比较采用多因素方差（Two-way ANOVA）分析；Kaplan-Meier生存曲线分析采用log-rank检验。P<0.05为差异有统计学意义。
2. 结果
2.1 构建LTβR-NIH3T3细胞系
利用慢病毒感染，在NIH3T3细胞中过表达LTβR，流式检测发现GFP+细胞比例达99%，转染效率高；LTβR-NIH3T3细胞相较于Vector-NIH3T3细胞，LTβR+细胞比例增加至近70%（图1A）。同时，qPCR进一步证明，流式分选能提高LTβR-NIH3T3细胞中过表达Ltbr水平（图1B），上述结果均表明过表达LTβR的NIH3T3细胞构建成功。
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图 1 检测LTβR-NIH3T3细胞中LTβR的过表达效率Figure 1. Examination of LTβR overexpression in LTβR-NIH3T3 cellsA: Flow cytometry analysis of the expression of LTβR in Vector-NIH3T3 and LTβR-NIH3T3 cells; B: qPCR analysis of the expression of Ltbr in Vector-NIH3T3 and LTβR-NIH3T3 cells including before and after sorting. ****P<0.000 1, n=3.2.2 过表达LTβR对NIH3T3细胞非经典NF-κB通路的激活作用
Western blot结果显示，相较于NIH3T3细胞和Vector-NIH3T3细胞，过表达 LTβR的NIH3T3细胞胞质内P100减少，细胞核内P52和RelB增加（图2）。qPCR结果显示，在未处理和anti-LTβR激活型抗体刺激24 h这两种条件下，与Vector-NIH3T3细胞相比，LTβR-NIH3T3细胞中趋化因子Ccl19、Ccl21的表达量均提高，差异有统计学意义（P<0.05），黏附因子Madcam1、Icam1和Vcam1的表达量亦增加（图3）。与未处理相比，anti-LTβR激活型抗体刺激24 h后，LTβR-NIH3T3细胞中Ccl19、Ccl21的表达增加，其中Ccl21的差异有统计学意义（P<0.05），黏附因子Madcam1、Icam1和Vcam1的表达量增加，差异有统计学意义（P<0.05）。说明在过表达LTβR的NIH3T3细胞中，非经典NF-κB信号通路被明显激活。
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图 2 Western blot检测非经典NF-κB通路在LTβR-NIH3T3细胞中的激活Figure 2. Western blot was used to examine the activation of non-canonical NF-κB pathway in LTβR-NIH3T3 cells.The relative molecular mass goes from top to bottom: 100×103, 44×103, 52×103, 70×103, 68×103.2.3 LTβR-NIH3T3细胞的抗肿瘤效果
与Vector-NIH3T3治疗组相比，皮下注射LTβR-NIH3T3细胞后有效抑制黑色素瘤增长，肿瘤体积更小，肿瘤质量更轻，荷瘤小鼠生存期延长，差异均有统计学意义（P<0.05，图4A～4C）。同时，LTβR-NIH3T3细胞治疗小鼠的肿瘤附近出现淋巴样组织，且肿瘤切片显示肿瘤炎性细胞浸润增加，说明TLO在肿瘤附近诱导成功（图4D）。
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图 3 qPCR检测非经典NF-κB通路在LTβR-NIH3T3细胞中的激活Figure 3. qPCR was done to examine the activation of non-canonical NF-κB pathway in LTβR-NIH3T3 cells*P<0.05,**P<0.01, ***P<0.001. Vec: Vector-NIH3T3 cells; LTβR: LTβR-NIH3T3 cells. n=3.![]()
图 4 LTβR-NIH3T3细胞的抗肿瘤效果评价Figure 4. Evaluation of antitumor function of LTβR-NIH3T3 cellsA: Measurement of tumor size (Vec: n=11; LTβR: n=15); B: The tumor was weighted at 21 d after tumor stripping (Vec: n=11; LTβR: n=15); C: The tumor growth curves (Vec: n=11; LTβR: n=15); D: HE staining of tumor microenvironment (white triangles show lymphoid-like tissue). Vec: Vector-NIH3T3 cells; LTβR: LTβR-NIH3T3 cells; ** P<0.01, Vec vs. LTβR.2.4 LTβR-NIH3T3细胞对免疫细胞浸润至肿瘤的促进作用
流式染色结果显示（图5），注射LTβR-NIH3T3细胞治疗后肿瘤免疫微环境中CD45+细胞浸润增加，其中CD3+T淋巴细胞（包括CD4+ T细胞和CD8+ T细胞）和CD11b+Ly6C-Ly6G-巨噬细胞占活细胞的比例和单位体积（每mm3肿瘤）中的细胞绝对数量均增加，提示LTβR-NIH3T3治疗的小鼠体内有更强的抗肿瘤免疫反应。
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图 5 LTβR-NIH3T3细胞影响肿瘤浸润免疫细胞类型Figure 5. Composition of tumor infiltrating immune cells affected by LTβR-NIH3T3 cellsA: Flow cytometry analysis of tumor infiltrating immune cells; B: Proportion of live cells; C: Total cell number/mm3 tumor. *P<0.05, **P<0.01. Vec: Vector-NIH3T3 cells (n=11); LTβR: LTβR-NIH3T3 cells (n=15).2.5 LTβR-NIH3T3细胞对巨噬细胞向MHCⅡ+促炎表型极化的促进作用
进一步分析LTβR-NIH3T3细胞治疗后肿瘤浸润的巨噬细胞发现，MHCⅡ+F4/80−巨噬细胞比例增加，单位体积（每mm3肿瘤）中的细胞绝对数量显著增加，MHCⅡ−F4/80+巨噬细胞比例和单位体积（每mm3肿瘤）中的细胞绝对数量均无明显变化（图6A，6B）；MHCⅡ+/(F4/80+)比例增加（图6C），提示LTβR-NIH3T3细胞通过促进巨噬细胞向MHCⅡ+促炎表型极化，在肿瘤微环境中增强抗原递呈，从而抑制肿瘤生长。
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图 6 LTβR-NIH3T3细胞影响肿瘤巨噬细胞类型Figure 6. The effect of LTβR-NIH3T3 cells on macrophages in tumorsA: Flow cytometry analysis of MHCII+F4/80－ macrophages and MHCⅡ－F4/80+ macrophages; Ba, Bb, Bc, Bd: Proportion and total cell number/mm3 tumor of MHCⅡ+F4/80－ macrophages and MHCⅡ－F4/80+ macrophages; C: Ratio of MHCⅡ+/(F4/80+). * P<0.05, **P<0.01. Vec: Vector-NIH3T3 cells (n=11); LTβR: LTβR-NIH3T3 cells (n=15).3. 讨论
基因突变的积累导致肿瘤抗原的表达，触发机体的固有免疫和适应性免疫[22]。针对肿瘤的适应性免疫通常发生在SLO中，肿瘤部位的DC细胞摄取肿瘤抗原后迁移到SLO，递呈给T细胞，引起T细胞增殖和分化为多种效应和记忆细胞类型，迁移至肿瘤部位发挥作用[23]。在多个肿瘤类型中，易位淋巴器官TLO的发现，补充了人们对于抗肿瘤适应性免疫的认识[12, 16, 24-25]。
TLO出现在非淋巴部位，其产生和维持依赖持续感染或慢性炎症环境[3]。LTβR是TNF超家族的成员之一，LTβR介导的非经典NF-κB信号通路在TLO的形成过程中发挥关键作用[26-27]。非经典NF-κB信号通路的中心事件是P100加工，P100是P52的前体，激活非经典NF-κB信号通路后，P100加工成P52并释放RelB，形成P52/RelB异二聚体，进入细胞核内调控靶基因转录，引起下游趋化因子的表达[5,27]。这些趋化因子参与淋巴细胞的招募、迁移和归巢[8, 11]。同时，TLO高表达多个趋化因子（CCL19，CCL21，CXCL12，CXCL13等），提示这些趋化因子可作为鉴定肿瘤部位是否存在TLO的标记物[24, 28-29]。本研究在过表达LTβR的NIH3T3细胞中观察到胞质中P100减少，P52/RelB入核增加；无论有无激活型抗体刺激，下游趋化因子（Ccl19，Ccl21）和黏附因子（Madcam1，Icam1，Vcam1）的表达在mRNA水平显著增加，说明LTβR-NIH3T3细胞中非经典NF-κB信号通路被激活，具有诱导形成TLO的潜能。
通过在肿瘤附近诱导TLO的形成，有助于募集各种免疫细胞发挥局部的抗肿瘤免疫反应。为研究LTβR-NIH3T3细胞是否能够在肿瘤附近诱导TLO的形成，我们构建了黑色素瘤小鼠模型，相较于Vector-NIH3T3细胞组，LTβR-NIH3T3细胞治疗后，荷瘤小鼠肿瘤体积更小，生存期延长，肿瘤附近出现淋巴样结构。HE染色显示肿瘤微环境中免疫细胞浸润增加，流式细胞术对这些免疫细胞类型进一步分析，T细胞和巨噬细胞占活细胞比例和单位体积（每mm3肿瘤）中的细胞绝对数量增加。巨噬细胞是一类高度可塑性的细胞，能够响应各种微环境信号，极化为具有不同特性和功能的异质性群体[30]。其中，MHCⅡ+巨噬细胞发挥高抗原递呈作用，在肿瘤治疗中与良好的预后相关[31]。而F4/80+巨噬细胞则支持血管生成以及表达免疫抑制分子，如程序性死亡配体1和转化生长因子β等，促进肿瘤生长[32-33]。本研究结果显示，LTβR-NIH3T3细胞处理后，作用于抗肿瘤的MHCⅡ+F4/80−巨噬细胞比例增加，MHCⅡ+/（F4/80+）比例升高。上述结果表明LTβR-NIH3T3细胞成功在黑色素瘤小鼠的肿瘤部位诱导形成TLO，并促进了免疫细胞向肿瘤部位浸润。LTβR-NIH3T3细胞通过促进巨噬细胞向MHCⅡ+促炎表型极化，增强肿瘤抗原的递呈，促进T细胞的抗原特异性活化，发挥杀伤癌细胞作用。此外，促炎表型巨噬细胞已被证实具有在病理情况下替代LTi细胞诱导TLO形成的能力[34]。通过将巨噬细胞重新极化为促炎表型或抑制肿瘤相关巨噬细胞的存活、增殖、转移等策略的药物开发已经应用于肿瘤治疗中[32]。
在肿瘤微环境持续的抗原暴露和/或炎症刺激下，CD8 T细胞被激活形成CD8+细胞毒性 T 淋巴细胞，是清除癌细胞最有力的免疫细胞类型。CD8+ T细胞的肿瘤浸润程度是免疫检查点阻断治疗结果的预测标志物。接下来，我们将联合抗程序性细胞死亡蛋白1治疗，验证TLO的形成能否影响肿瘤浸润免疫细胞尤其是耗竭T细胞的命运从而促进抗肿瘤免疫。
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利益冲突 所有作者均声明不存在利益冲突
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图 1 检测LTβR-NIH3T3细胞中LTβR的过表达效率
Figure 1. Examination of LTβR overexpression in LTβR-NIH3T3 cells
A: Flow cytometry analysis of the expression of LTβR in Vector-NIH3T3 and LTβR-NIH3T3 cells; B: qPCR analysis of the expression of Ltbr in Vector-NIH3T3 and LTβR-NIH3T3 cells including before and after sorting. ****P<0.000 1, n=3.
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图 2 Western blot检测非经典NF-κB通路在LTβR-NIH3T3细胞中的激活
Figure 2. Western blot was used to examine the activation of non-canonical NF-κB pathway in LTβR-NIH3T3 cells.
The relative molecular mass goes from top to bottom: 100×103, 44×103, 52×103, 70×103, 68×103.
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图 3 qPCR检测非经典NF-κB通路在LTβR-NIH3T3细胞中的激活
Figure 3. qPCR was done to examine the activation of non-canonical NF-κB pathway in LTβR-NIH3T3 cells
*P<0.05,**P<0.01, ***P<0.001. Vec: Vector-NIH3T3 cells; LTβR: LTβR-NIH3T3 cells. n=3.
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图 4 LTβR-NIH3T3细胞的抗肿瘤效果评价
Figure 4. Evaluation of antitumor function of LTβR-NIH3T3 cells
A: Measurement of tumor size (Vec: n=11; LTβR: n=15); B: The tumor was weighted at 21 d after tumor stripping (Vec: n=11; LTβR: n=15); C: The tumor growth curves (Vec: n=11; LTβR: n=15); D: HE staining of tumor microenvironment (white triangles show lymphoid-like tissue). Vec: Vector-NIH3T3 cells; LTβR: LTβR-NIH3T3 cells; ** P<0.01, Vec vs. LTβR.
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图 5 LTβR-NIH3T3细胞影响肿瘤浸润免疫细胞类型
Figure 5. Composition of tumor infiltrating immune cells affected by LTβR-NIH3T3 cells
A: Flow cytometry analysis of tumor infiltrating immune cells; B: Proportion of live cells; C: Total cell number/mm3 tumor. *P<0.05, **P<0.01. Vec: Vector-NIH3T3 cells (n=11); LTβR: LTβR-NIH3T3 cells (n=15).
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图 6 LTβR-NIH3T3细胞影响肿瘤巨噬细胞类型
Figure 6. The effect of LTβR-NIH3T3 cells on macrophages in tumors
A: Flow cytometry analysis of MHCII+F4/80－ macrophages and MHCⅡ－F4/80+ macrophages; Ba, Bb, Bc, Bd: Proportion and total cell number/mm3 tumor of MHCⅡ+F4/80－ macrophages and MHCⅡ－F4/80+ macrophages; C: Ratio of MHCⅡ+/(F4/80+). * P<0.05, **P<0.01. Vec: Vector-NIH3T3 cells (n=11); LTβR: LTβR-NIH3T3 cells (n=15).
表 1 qPCR引物序列及长度
Table 1 Sequences and length of qPCR primers
Gene Forward (5′→3′) Reverse (5′→3′) Length/bp Ltbr CATGCTAGCATGCGCCTGCCCCGGGCCTC TGAGCGGCCGCTCAGAGGTCTTGGCATCCTAGTG 212 Ccl19 GAAAGCCTTCCGCTACCTTC GAGGTGCACAGAGCTGATAG 92 Ccl21 TCCGAGGCTATAGGAAGCAA CTTCCTCAGGGTTTGCACAT 108 Madcam1 GAGCAAGAAGAGGAGATACAAGAG TGGTGACCTGGCAGTGAAG 117 Icam1 GTGCTTTGAGAACTGTGGCA GGTCCTTGCCTACTTGCTG 119 Vcam1 GGAAGCTGGAACGAAGTATCC AAACACTTGACCGTGACCG 109 18S ACAGGGAGAAAGCGCAAAAC TGTGGCCTTGTGGTGAAGAG 237 
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