恶性肿瘤胸腹水中TIL生物学活性研究

从卵巢癌腹水和肺癌胸水中分离出肿瘤浸润淋巴细胞（ＴＩＬ），用ｒＩＬ－２诱导培养，观察其增殖活性。同时观察了ＰＪ－ＣＷ、ＰＯＧＰ、ＰＨＡ等对其杀伤活性的调节作用。结果表明，在培养５～１０天时ＴＩＬ增殖较快，其表面产生的ＣＤ３、ＣＤ４、ＣＤ８、Ｔａｃ等抗原，随培养时间的延长，表达量增加，尤以ＣＤ８增加较明显，ＣＤ４／ＣＤ８比值逐降。其表面产生的ＨＬＡ－ＤＲ、ＨＬＡ－ＡＢＣ抗原，在培养１５天内有增加表现，以后下降。其杀伤活性在培养５～１０天较高，其后活性下降。在培养系统中加入ＰＪ－ＣＷ、ＰＯＧＰ、ＰＨＡ等后，培养５天时，ＴＩＬ杀伤活性分别为８２．７０％，８５．５０％，８４．３５％（对照组为内保持较高的杀伤活性。     

肿瘤浸润淋巴细胞生物学性状初步研究

本研究发现，掺杂肿瘤细胞的早期ＴＩＬ培养上清液抑制自身淋巴结来源的淋巴细胞增殖作用；后期时，掺杂肿瘤细胞消失（即ＴＩＬ呈对数生长期），ＴＩＬ培养上清液却对自身淋巴结来源的淋巴细胞呈现生长刺激现象。首次发现消化酶对ＴＩＬ这些活力指标有影响。表型与淋巴细胞形态学对照研究时发现，初次分离的ＴＩＬ层细胞中，Ｇｉｅｍ－ｓａ染色的淋巴细胞比例为８３±６％；活细胞镜下计数淋巴细胞占７３±１２％；而ＦＡＣＳ分析ＣＤ３只有２３±８％，ＨＬＡＤＲ３１±２０％；是否属ＴＩＬ前体细胞，还须进一步研究。     

恶性脑肿瘤浸润淋巴细胞的临床应用研究

用冷酶法对７例脑恶性肿瘤进行肿瘤浸润淋巴细胞分离、培养并用于临床。７例肿瘤的ＴＩＬ扩增倍数均大于刚分离时的５００倍，其中２例大于１０００倍，另２例分别大于２０００及３０００倍；ＴＩＬ的表型检测ＣＤ３、ＣＤ４、ＣＤ８、ＨＬＡ－ＤＲ分别为９７％，３５％，５４％，９７％；电镜观察到ＴＩＬ先靠近肿瘤细胞，伸出触手，接触肿瘤细胞膜上，随后肿瘤细胞破坏、死亡的过程。临床应用ＴＩＬ后随访至今９ｍｏ，７例肿瘤患者     

rIL-2对大肠癌TIL体外培养的影响

将大肠癌ＴＩＬ在含有ｒＩＬ-２浓度为６０、６００和６００Ｕ／ｍｌ的培养基中分别培养扩增，观察不同浓度ｒＩＬ-２对ＴＩＬ增殖力、杀伤活性、表型的影响。结果表明：低浓度ｒＩＬ-２组较中、高浓度组增殖力减弱，但对自身肿瘤细胞的杀伤活性较中、高浓度组增高。三组ＴＩＬ培养后ＣＤ３＋细胞比例显著增加，ＣＤ４＋细胞比例无明显变化，低、中浓度组ＣＤ８＋细胞比例显著增加，低浓度组ＣＤ２５＋细胞比例显著增加。说明低浓度ｒＩＬ-２可选择性刺激ＣＤ８＋、ＣＤ２５＋Ｔ细胞克隆增殖，使大肠癌ＴＩＬ对自身肿瘤杀伤活性提高。     

恶性肿瘤胸腺水中TIL生物学活性研究

从卵巢癌腹水和肺癌胸水中分离出肿瘤浸润淋巴细胞，用ｒＩＬ－２诱导培养，观察其增殖活性。同时观察了ＰＪ－ＣＷ，ＰＯＧＰ，ＰＨＡ等对其杀伤活性的调节作用。结果表明，在培养５－１０天时ＴＩＬ增殖较快，其表面产生的ＣＤ３，ＣＤ４，ＣＤ８，Ｔａｃ等抗原，随培养时间的处长，表达量增加，尤以ＣＤ８增加较明显，ＣＤ４／ＣＤ８比值逐降。     

狼疮性肾炎患者淋巴细胞亚群比率和免疫球蛋白水平的变化

目的 探讨狼疮性肾炎（ＬＮ）患者的淋巴细胞亚群和免疫球蛋白（Ｉｇ）的变化及其意义。方法采用淋巴细胞的膜抗原双标记染色法及ＥＬＩＳＡ，对６０例ＬＮ患者的细胞与体液免疫功能进行前瞻性研究。结果①与对照组相比较，活动期ＬＮ患者的ＣＤ３＋ＣＤ４＋细胞的比率（Ａ组：伴肾病综合征者）为（ｌ６．３ ±７．９）％，Ｂ组（不伴肾病综合征者）为（２０．８±１０．１）％］和ＣＤ１６＋＋ＣＤ５６＋细胞的比率［Ａ组为（８．６±５．７）％，Ｂ组为（１５．２±６．２）％明显减少；ＣＤ３+ ＣＤ8+”细胞的比率［Ａ组为（４８．３±１０．７）％，Ｂ组为（３５．９±９．８）％］明显增高；ＣＤ３+ ＣＤ４+ ／ＣＤ３+ ＣＤ８＋细胞的比值＜１（Ａ组为０．４３±０．２１，Ｂ组为０．８７±０．２５），且Ａ组与８组相比较差异明显（Ｐ＜０．０５）。②与活动期ＬＮ患者相比较，治疗后处于稳定期的 ＬＮ患者 ＣＤ３＋ＣＤ４＋细胞的比率卜组为（２８．８±８．ｌ）％，Ｂ组为（３２．８± ７．ｌ）％］和 ＣＤ１６＋＋ＣＤ５６＋细胞的比率［Ａ组为（１８．９ ± １２．５）％，Ｂ组为（２４．０±８． ９）％，以及 ＣＤ３＋ＣＤ４＋／ＣＤ３＋ＣＤ８＋细胞的比值（Ａ组为 ０．９７±２．３，Ｂ组     

MHC-Ⅰ类分子在肿瘤浸润性淋巴细胞识别人黑色素瘤细胞中的作用

应用人白细胞抗原－Ａ２阳性（ＨＬＡ－Ａ＋２）黑色素瘤病人的瘤块中分离得到的肿瘤浸润性淋巴细胞（ＴＩＬ），进行ＨＬＡ－Ａ２限制性ＴＩＬ抗人黑色素瘤细胞作用的研究。在实验中发现，ＴＩＬ对自身和同种异体的ＨＬＡ－Ａ＋２黑色素瘤细胞具有杀伤作用，而对ＨＬＡ－Ａ－２黑色素瘤细胞及ＨＬＡ－Ａ＋２非黑色素瘤细胞无作用。选择重组白细胞介素４（ｒＩＬ－４）＋肿瘤坏死因子α（ＴＮＦα）能促进黑色素瘤细胞ＨＬＡ－Ａ２表达量，并增强ＴＩＬ对瘤细胞的杀伤活性。抗ＣＤ３、抗ＨＬＡ－ＡＢＣ和抗ＨＬＡ－Ａ２单抗具有明显抑制ＴＩＬ的抗瘤细胞活性。结果表明ＴＩＬ杀伤黑色素瘤细胞依赖于Ｔ细胞受体（ＴＣＲ）对瘤细胞共同抗原的识别，并有主要组织相容性复合体－Ⅰ类分子参与。     

HLA多态性与HIV感染及AIDS发病相关性的研究

为研究人类白细胞抗原（ＨＬＡ）和人免疫缺陷病毒（ＨＩＶ）易感性的关系，本文分析比较了以下三组人群的ＨＬＡ表型频率和单倍型频率：①１７２例正常人；②１７例血清ＨＩＶ阴性高危人群；③１８０例血清ＨＩＶ阳性患者，其中２１例发展为艾滋病（ＡＩＤＳ），３７例６个月内ＣＤ４＋淋巴细胞降低至少２０％（ＣＤ４ｄｅｃｌｉｎｅ）。在１７２例对照和１８０例血清ＨＩＶ阳性受试者的比较中发现，其ＨＬＡ表型频率和单倍型频率没有显著差别，提示ＨＩＶ感染与ＨＬＡ无关联。然而，ＨＬＡ－Ｂ２１、ＨＬＡ－Ｂ８、ＨＬＡ－Ｂ３５表型及ＨＬＡ－Ａ１－Ｂ８、ＨＬＡ－Ｂ８－ＤＲ３、ＨＬＡ－Ａ１－Ｂ８－ＤＲ３单倍型与ＣＤ４阳性淋巴细胞下降，或与血清ＨＩＶ感染发展成ＡＩＤＳ病显著相关。１７例ＨＩＶ血清阴性高危人群中，无一携带单倍型ＨＬＡ－Ａ１－Ｂ８－ＤＲ３，提示该单倍型可能与ＨＩＶ感染的抗性相关。     

T细胞表面6种细胞表型的变化与大型癌分期及术式的关系

应用流式细胞仪检测３９例大肠癌患者手术前后Ｔ细胞表面６种细胞表型。发现随着Ｄｕｋｅｓ分期的增高，术前ＣＤ３，ＣＤ４，ＣＤ４／ＣＤ８，ＣＤ１６，ＣＤ６９及ＣＤ＾３＋／ＨＬＡ－ＤＲ＾＋下降，ＣＤ８逐渐增高；Ａ期大肠癌患者机体免疫功能活跃，术前ＣＤ３，ＣＤ４高于对照组，ＣＤ８，ＣＤ４／ＣＤ８，ＣＤ１６，ＣＤ＾＋３／ＨＬＡ－ＤＲ＾＋与对照组相同．     

肺鳞癌肿瘤浸润淋巴细胞的体外扩增,抗瘤活性及表型分析

对８例人原发性肺鳞癌的肿瘤浸润淋巴细胞（ＴＩＬ）进行分离培养并检测了ＴＩＬ的抗瘤活性及细胞表型，结果表明８例ＴＩＬ的平均扩增倍数为４３０；ＴＩＬ在１０～３０天之间显示出高水平的非特异性杀伤活性；１０天～时对Ａ１及Ｄａｕｄｉ细胞的杀伤百分率分别为７９．２％和４３．４％，２０天～时分别为５１．７％和２７．９％。非特异杀伤活性在３０天以后降至较低水平，所进行的几次实验表明肺鳞癌ＴＩＬ对自体癌细胞的杀伤活     

T淋巴细胞清除促进脐血造血细胞的体外扩增

采用抗CD3或抗CD8单克隆抗体的补体细胞毒方法清除脐血单个核细胞 (MNC )中T淋巴细胞 ,CD34免疫亲和柱纯化MNC中CD34 +细胞 ,流式细胞技术 (FACS )分析细胞表面标志。将CD34 +细胞中加入含多种造血生长因子的培养基进行体外液态扩增 ,并观察粒 巨噬集落 (CFU GM )和多向祖细胞集落 (CFU GEMM )形成能力。结果CD3+或CD8+细胞清除组和MNC经CD34免疫亲和柱纯化后 ,CD34 +细胞分别为 5 9 5 2 %、 5 6 70 %和 5 0 72 % ,比未纯化组 (1 0 7% )纯度大幅度提高。抗CD3单抗清除 +CD34纯化组和单纯CD34纯化组经造血生长因子刺激培养第 14天 ,细胞总数分别扩增 110 40倍和 87 0 0倍。抗CD3单抗清除 +CD34纯化组、抗CD8单抗清除 +CD34纯化组和单纯CD34纯化组的CFU GM产率分别为 2 86 5 0± 12 0 2、2 88 5 0± 17 68和 2 19 5 0± 5 3 0 3,前者虽高于后者 ,但差异无显著性。CFU GEMM产率分别为 376 67± 43 2 4、 438 33± 36 73和 311 0 0± 40 11,抗CD3单抗清除 +CD34纯化组无显著差异 ,抗CD8单抗清除 +CD34纯化组显示出显著性作用 (P <0 0 5 )。     

Tumor-infiltrating lymphocyte subsets and tertiary lymphoid structures in pulmonary metastases from colorectal cancer

The presence of tumor-infiltrating lymphocytes (TILs) and tertiary lymphoid structures (TLSs) reflects an active inflammatory tumor microenvironment. High density of TILs as well as presence of TLS is associated with improved survival in various solid cancer types. We aimed to describe the density and distribution of TILs and TLS in pulmonary metastases (PMs) from primary colorectal cancer (CRC) and its correlation with clinicopathological variables. Fifty-seven CRC pulmonary metastasectomy specimen (PM) and 31 matched primary CRC specimen were included. Cluster of differentiation (CD)3+, CD8+, CD45RO+ and FoxP3+ TILs were evaluated by immunohistochemistry and density was scored semiquantitatively. TLS were evaluated based on morphological criteria. Survival time was defined from pulmonary metastasectomy to death or last follow up. A marked infiltration with CD3+, CD8+, CD45RO+ and FoxP3+ TILs was evident in CRC PM and matched primary CRC. Further assessment of the immune infiltrate in PM showed that a high density of FOXP3+ TILs at the invasive margin [HR 2.40 (1.11–6.96); P = 0.031] and low density of CD8+ cells in TLS [HR 0.30 (0.14–0.79); P = 0.016] were associated with a worse prognosis in univariate analysis. Moreover, a low CD8/FoxP3-ratio of TILs at the invasive margin (P = 0.042) and in TLS (P = 0.027) conferred an impaired prognosis after pulmonary metastasectomy. Our findings suggest that CRC PM harbor an immune active microenvironment. The balance of CD8+ and FoxP3+ T-cells at the tumor border and in TLS provides prognostic information in patients with CRC PM.     

Immunophenotype of tumor-infiltrating lymphocytes in atypical Spitzoid tumors according to the risk of progression

The aims of the study were to investigate and compare the immunophenotype of tumor-infiltrating lymphocytes (TILs) and PD-L1 expression in a series of benign, intermediate and malignant Spitzoid lesions showing marked inflammatory lymphoid component, to find out its possible relation with the prognosis of these lesions.Six out of 97 Spitz nevus (SN) (6 %), five out of 26 atypical Spitz tumors (AST) (16 %) and seven out of 37 Spitzoid melanomas (SM) (19 %) showed diffuse, intense inflammatory component and were included in the study. The biological risk of the tumors was assessed in all AST through the melanoma 4 probe-FISH assay and the 9p21 locus exploration. TILs were quantitatively immunophenotyped using CD3, CD4, CD8, CD20, TIA1, FOXP3 and PD1 antibodies. PD-L1 was assessed in tumoral cells and inflammatory cells adjacent to the tumor.No significant differences of TILs immunophenotype were found between SN, AST and SM. However, the classification of tumors according to the biological risk showed that grouped SN plus low-risk AST had a significantly higher number of T-cells CD8+ and TIA-1+, as well as a lower CD4/CD8 relation and B- lymphocyte number than high-risk of progression tumors (grouped high-risk AST plus SM). Immunoregulatory T-cell markers PD1 and FOXP3 only correlated with each other and with PD-L1 expression.In conclusion, The TILs immunoprofile differences between low-risk and high-risk of progression Spitzoid tumors, especially regarding CD8 and the cytotoxic immune response, can add prognostic information about these challenging tumors and impact the clinical management of patients.     

Down-Regulation of CD25 Expression on the Surface of Activated Tumor-Infiltrating Lymphocytes in Human Cervical Carcinoma

ABSTRACT: To investigate the activation status of tumor-infiltrating lymphocytes (TILs) within the tumor milieu of human cervical carcinoma, we quantitatively measured and compared the activation markers on lymphocyte subpopulations which infiltrating normal and neoplastic cervix. A total of 20 patients with stage IA to IIA cervical cancer (cancer group) and 10 women with normal cervix (control group) were enrolled in this study. Mononuclear cells were isolated from tissue specimens by mechanical dispersal technique and three-color flow cytometry was utilized for the quantification of activation markers on lymphocyte subsets. Compared with control group, lymphocytes isolated from cancer tissue consisted of higher proportions of B cells (7.23% ± 4.49% vs. 3.67% ± 3.19%, P = 0.016) and T cells (72.33% ± 8.70% vs. 53.15% ± 17.36%, P = 0.004), but an inverted CD4:CD8 ratio (0.74 ± 0.27 vs. 1.14 ± 0.28, P = 0.002) and decreased NK cells (7.53% ± 4.33% vs. 16.00% ± 11.82%, P = 0.035). Low expression of CD25, but not CD69 and HLA-DR was observed on both CD4⁺CD3⁺ and CD8⁺CD3⁺ T cells derived from cervical cancer (P < 0.0001). Further dual activation marker analysis demonstrated that the expression of CD25 was dissociated from CD69 and HLA-DR on the same TILs in cancer tissue (P < 0.001). TILs in the tumor microenvironment can be functionally inhibited and lose the ability of clonal proliferation due to depressed expression of CD25.     

Expression of Inhibitory Receptors in Natural Killer (CD3CD56) Cells and CD3CD56 Cells in the Peripheral Blood Lymphocytes and Tumor Infiltrating Lymphocytes in Patients with Primary Hepatocellular Carcinoma

The cytolytic responses of NK (CD3(-)CD56(+)) and CD3(+)CD56(+) cells are inhibited by the engagement of the killer inhibitory receptors (p58.1, p58.2, and CD94) with respective ligands on the target cell. The expression of these receptors in peripheral blood lymphocytes (PBLs) (n = 18) and tumor infiltrating lymphocytes (TILs) (n = 7) was examined in patients with primary hepatocellular carcinoma (HCC). There were no differences in the expression of the three inhibitory receptors by both NK and CD3(+)CD56(+) PBLs in patients with HCC compared to that of control NK and CD3(+)CD56(+) PBLs, respectively (all P = NS). However, the expression of p58.1 by NK TILs and by CD3(+)CD56(+) TILs in patients with HCC was significantly decreased compared to that of hepatic lymphocytes of the control subjects (8.9% vs 37.85%, P = 0.047; 4.1% vs 25.2%, P = 0.049, respectively). The expression of p58.2 by CD3(+)CD56(+) TILs and CD94 by NK TILs was also decreased compared to that of hepatic lymphocytes of the control subjects (16.9% vs 73.1%, P = 0.047; 21% vs 49.95%, P = 0.037, respectively). These changes were limited to hepatic TILs, and this observation may reflect an adaptive anti-tumor phenomenon occurring in the microenvironment of HCC.     

Inverse Relation of Fas-Ligand and Tumor-Infiltrating Lymphocytes in Angiosarcoma : Indications of Apoptotic Tumor Counterattack

Fas and Fas-L regulate immune responses through the induction of cell death. Fas-L is commonly expressed in activated immune cells and in the endothelium. In the latter it contributes to the inhibition of transvascular cell migration by the induction of apoptosis in Fas-bearing lymphocytes. Here we investigated whether the Fas/Fas-L system may regulate lymphocyte invasion into angiosarcomas. Fas and Fas-L expression was quantitatively determined in different grade angiosarcomas (n = 40) and related to the number of extravasated tumor-infiltrating lymphocytes (TILs). Fas expression was detected in < 50% of the cases. In positive tumors both the number of Fas-positive cells and the staining intensity were highly variable and did not correlate with the number of TILs, the mean time of survival, and the histopathological tumor grade. By contrast, Fas-L expression was detected in >70% of the cases and the relative numbers of Fas-L-positive cells correlated inversely with the numbers of CD3- and CD8-positive TILs (P < or = 0.004). The survival times of patients with high Fas-L-expressing angiosarcomas were significantly reduced as compared to patients with low Fas-L-expressing tumors. Our results show that angiosarcomas with low Fas-L expression are characterized by numerous TILs, whereas sarcomas with high Fas-L expression show significantly reduced numbers of TILs. These results suggest that the Fas/Fas-L system may repress TIL invasion into angiosarcoma and by this may contribute to the evasion of the anti-tumor immune surveillance of angiosarcoma in the course of an apoptotic tumor counterattack mechanism.     

3TSR治疗裸鼠胃癌的实验研究

目的： 探讨血管形成抑制剂3TSR对人胃癌裸鼠皮下移植瘤的抑制作用及机制。方法: 建立人胃癌裸鼠皮下种植瘤模型,实验分为对照组(腹腔注射PBS 0.2 mL)、3TSR组(腹腔注射3TSR 3 mg/kg BW)，每组8只。给药3周后处死动物,提取瘤体，测量各组荷瘤体积，HE染色检测肿瘤坏死面积百分比，免疫组织化学方法检测肿瘤微血管表达、肿瘤细胞增殖指数，原位末端标记法(TUNEL法)检测肿瘤细胞凋亡率,CD31/TUNEL/DAPI重复染色法测定肿瘤血管内皮细胞凋亡率。结果: 3TSR组平均肿瘤体积为(648.34±126.90) mm3,较对照组显著缩小(P<0.05)； 3TSR组平均肿瘤坏死面积百分比为（39.6±7.8）%,较对照组扩大69.2%，2组间有显著差异(P<0.05)。3TSR组肿瘤平均微血管数为12.8±4.1,平均微血管面积为（689.3±118.6）μm2，均显著低于对照组(P<0.05)，3TSR组增殖指数和凋亡率分别为(40.0±7.1)%和(3.4±1.2)%，与对照组比较无显著差异(P>0.05),3TSR组肿瘤血管内皮细胞凋亡率为(11.6±2.8)%,显著高于对照组的(2.9±1.5)%,P<0.05。结论: 3TSR能抑制裸鼠胃癌新生血管形成，具有显著减少肿瘤体积、肿瘤血管和增加肿瘤细胞坏死的作用，但对胃癌细胞无直接抑制作用，其机制可能与诱导内皮细胞凋亡有关。     

Expansion and immunological study of human tumor infiltrating gamma/delta T lymphocytes in vitro.

Goffa/delta T cells have stimulated a lot of interest because of their unique features in antigen recognition and cytotoxicities to many autologous and/or allogeneic tumor cells. We have developed a novel method to selectively expand larger amounts of human tumor-infiltrating gamma/delta T lymphocytes (gamma/delta TILs) ex vivo by immobilized pan- anti-TCRgamma/delta monoclonal antibody in the presence of exogenous IL-2. The expanded gamma/delta TILs mainly expressed CD45RO and HLA-DR molecules and did not express CD4. CD8+ gamma/delta TILs accounted for 19% of gamma/delta TILs. The expression of CD25 molecule on expanded gamma/delta T cells was inducible and downregulated following a time course. The Vdelta1 and Vdelta2 subsets amount to 37 and 58%, respectively. The expanded gamma/delta TILs show an IL-2-dependent proliferation, MHC class I-unrestricted and TCRgamma/delta-related cytotoxicities to two MHC class I+ and two MHC class I+ allogeneic tumor cell lines in vitro.