癸酸能够活化CD8+ T细胞并提高其抗肿瘤免疫反应能力

目的：探究中链脂肪酸癸酸对CD8+ T细胞活化的影响,及其对CD8+ T细胞介导的抗肿瘤免疫反应的作用和机制。方法：建立C57BL/6小鼠黑色素瘤B16F10 皮下荷瘤模型,随机分为癸酸组（10 mg/kg 癸酸灌胃）和对照组（等量溶剂灌胃）,观察癸酸对小鼠肿瘤生长以及生存率的影响,采用流式细胞术检测肿瘤微环境中浸润CD8+ T细胞的活化水平。建立B16F10-OVA和OT-I T细胞共培养体系,采用流式细胞术检测癸酸对CD8+ T细胞的肿瘤细胞杀伤能力的影响。采用α-CD8抗体清除B16F10 荷瘤小鼠体内CD8+ T细胞,观察对小鼠肿瘤体积的影响。小鼠原代CD8+ T细胞经癸酸处理后,采用WB、ELISA及qPCR、流式细胞术检测T细胞受体（TCR）活化、效应细胞因子产生以及增殖和代谢水平。在B16F10荷瘤小鼠模型中,观察α-PD-1抗体联合癸酸给药对小鼠肿瘤生长以及生存率的影响。结果：在小鼠黑色素瘤荷瘤模型中,与对照组相比,癸酸组小鼠移植瘤体积显著降低且生存率显著提高（均P<0.05）,肿瘤浸润CD8+ T细胞IFN-γ和TNF-α的表达水平显著升高（P<0.01）。经癸酸处理的OT-I T细胞对B16F10-OVA细胞的杀伤水平显著升高（P<0.01）。在荷瘤小鼠模型中用α-CD8 抗体清除CD8+ T 细胞后,癸酸对移植瘤的抑制作用显著降低（P<0.000 1）。小鼠原代CD8+ T细胞经癸酸处理后,TCR活化水平显著升高、细胞因子IL-2和IFN-γ的产生增多、线粒体代谢水平显著上调（均P<0.05）。在黑色素瘤荷瘤小鼠模型中,癸酸与α-PD-1抗体联用,能够显著抑制小鼠移植瘤生长并提高其生存率（均P<0.05）。结论：癸酸能够促进CD8+ T细胞活化、增强其抗肿瘤免疫反应能力。     

肺癌细胞外泌体miR-24-3p调控CD8+T细胞抗肿瘤的功能及机制研究

目的:探究肺癌细胞外泌体miR-24-3p调控CD8+T细胞抗肿瘤的功能及机制。方法:将16HBE exo、A549 exo、H522 exo、H460 exo、miR-NC exo、miR-24-3p exo与CD8+T细胞共孵育（16HBE exo组、A549 exo组、H522 exo组、H460 exo组、miR-NC exo组、miR-24-3p exo组），与PBS共孵育组（PBS组）作为对照，CD8+T与miR-24-3p exo共孵育后转染FGF11质粒（miR-24-3p exo+FGF11组）。CCK8法检测CD8+T细胞的增殖能力。双荧光素酶报告基因实验验证miR-24-3p和FGF11的靶向关系。将各组CD8+T细胞与A549细胞以20∶1的比例共孵育4 h（16HBE exo/CD8+T组、A549 exo/CD8+T组、H522 exo/CD8+T组、H460 exo/CD8+T组、miR-NC exo/CD8+T组、miR-24-3p exo/CD8+T组、PBS/CD8+T组、miR-24-3p exo+FGF11/CD8+T组），Elisa检测共孵育后细胞上清中INF-γ和IL-2的浓度，LDH法检测CD8+T细胞对A549细胞的杀伤能力。结果:相比于PBS组，A549 exo组、H522 exo组、H460 exo组CD8+T细胞增殖能力显著降低（P＜0.05）。A549 exo/CD8+T组、H522 exo/CD8+T组、H460 exo/CD8+T组细胞上清中INF-γ和IL-2的浓度显著下降（P＜0.001），CD8+T细胞杀伤能力亦显著下降（P＜0.001）。双荧光素报告基因检测结果显示FGF11为miR-24-3p的靶基因。miR-24-3p exo组CD8+T细胞增殖能力显著低于miR-NC exo组，miR-24-3p exo+FGF11组CD8+T细胞增殖能力显著高于miR-24-3p exo组（P＜0.05）。miR-24-3p exo/CD8+T组细胞上清中INF-γ和IL-2浓度及CD8+T细胞杀伤能力均显著低于miR-NC exo/CD8+T组（P＜0.001）， miR-24-3p exo+FGF11/CD8+T组细胞上清中INF-γ和IL-2浓度及CD8+T细胞杀伤能力显著高于miR-24-3p exo/CD8+T组（P＜0.001）。结论:肺癌细胞外泌体miR-24-3p可通过靶向抑制FGF11而抑制CD8+T细胞的抗肿瘤功能。     

DC-CIK对胃癌合并腹水患者外周血CD4+CD25+调节性T细胞增殖及功能的影响

目的:探讨DC-CIK对胃癌合并腹水患者外周血CD4+CD25+调节性T胞(Treg细胞)比例及功能的影响。方法:60例胃癌合并腹水患者,于输注DC-CIK前1天及DC-CIK治疗结束后1周分别采集外周血。流式细胞术检测外周血Treg细胞的比例,RT-PCR法检测其Foxp3mRNA表达情况;将分选出的Treg细胞和CD4+CD25-T细胞分为单纯Treg细胞组（A组）、1∶1混合培养（B组）、单纯CD4+CD25-细胞组（C组）进行培养,3H-TdR掺入法检测Treg细胞抑制CD4+CD25-细胞增殖的能力。结果:治疗后外周血Treg细胞占CD4+T细胞的比例较疗前显著下降［(6.21±1.37)% vs (9.38±1.06)%,P＜0.05］。治疗后Treg细胞Foxp3mRNA表达水平较治疗前显著下降［(56.18±13.25)% vs (85.26±11.58)%,P＜0.05］。治疗后Treg对CD4+CD25-T细胞抑制增殖能力较治疗前明显下降［(37.31±4.16)% vs (48.92±5.25)%,P＜0.05］。结论:输注DC-CIK免疫治疗,可显著降低胃癌合并腹水患者外周血Treg细胞比例,下调Foxp3mRNA表达水平,降低Treg细胞免疫抑制功能,有利于诱导抗肿瘤免疫效应。     

聚肌胞对活化早期T细胞PD-1表达的诱导及对黑色素瘤生长的抑制作用

 目的 探讨活化早期阶段（脾组织内，未向靶器官迁移的）T细胞程序性死亡受体1（PD-1）表达情况。方法 6~8周龄C57BL/6小鼠随机分组后，以Poly I:C肽疫苗免疫，7天后获取小鼠脾细胞，观察Poly I:C对非特异性及特异性T细胞活化的影响，并检测特异性CD8⁺T细胞表面PD-1表达。同时，通过敲除CD8细胞观察Poly I:C的抗肿瘤效应是否与CD8⁺T细胞相关。结果 小鼠经Poly I:C免疫后，脾组织内非特异性及特异性T细胞活化均显著增强；活化的特异性CD8⁺T细胞虽高表达PD-1，但仍可合成T细胞功能性分子IFN-γ。此外，Poly I:C能显著抑制荷瘤小鼠黑色素瘤生长（P=0.0243），且该效应与CD8+T细胞有关。结论 Poly I:C可以促进T细胞活化；活化早期的（脾组织内）T细胞虽高表达PD-1，但仍具有功能。     

CD3+CD56+NKT细胞及CD3-CD56+NK细胞在恶性肿瘤患者的临床意义研究

背景与目的细胞毒性淋巴细胞在抗肿瘤免疫效应中发挥着重要作用,CD3 CD56 NKT细胞作为一类新的具有细胞毒性的效应细胞,目前关于其抗肿瘤意义的探讨主要集中于血液系统恶性疾病,而在实体肿瘤中的应用和临床价值研究尚少。本研究旨在初步探讨抗肿瘤细胞CD3 CD56 NKT细胞及CD3-CD56 NK细胞在恶性肿瘤患者外周血的表达状态及其临床意义。方法采用流式细胞术分析118例恶性肿瘤患者(55例肺癌患者和63例乳腺癌患者)及46例健康对照组外周血中的T细胞亚群及CD3 CD56 NKT细胞、CD3-CD56 NK细胞表达。结果恶性肿瘤患者组CD3 CD8 T细胞、CD3 CD56 NKT细胞以及CD3-CD56 NK细胞表达率均明显高于健康对照组(P<0.01)。肺癌患者中以CD3 CD8 T细胞和CD3 CD56 NKT细胞明显增加为主;乳腺癌患者中以CD3 CD56 NKT细胞和CD3-CD56 NK细胞明显增加为主。结论CD3 CD56 NKT细胞在肺癌和乳腺癌患者的抗肿瘤效应中占据重要地位,而CD3 CD8 CTL和CD3-CD56 NK细胞在不同类型肿瘤患者中具有不同的重要性。     

CD4+CD25+调节性T细胞在Lewis肺癌移植鼠中的检测及临床意义

目的:研究Lewis肺癌移植鼠胸腺与脾脏CD4 CD25 调节性T细胞数量及其相关基因Foxp3 mR-NA的表达特点,探讨CD4 CD25 调节性T细胞在诱导肿瘤免疫耐受中的作用。方法:将传代培养的Lewis肺癌细胞接种于C57BL/6小鼠右腋皮下,建立Lewis肺癌模型。观测成瘤率、平均瘤重、肿瘤体积动态变化;采用MTT法检测胸腺及脾脏T淋巴细胞增殖功能;采用流式细胞术检测胸腺及脾脏CD4 CD25 调节性T细胞数量变化;采用半定量RT-PCR方法检测肺癌小鼠胸腺与脾脏Foxp3 mRNA表达水平。结果:Lewis肺癌的移植成瘤率为100%,平均瘤重(3.34±1.79)g;胸腺T淋巴细胞增殖功能略有降低,但降低不显著(P>0.05),而脾脏T淋巴细胞增殖功能明显降低(P<0.05);胸腺及脾脏CD4 CD25 调节性T细胞数量均明显升高(P<0.05);Foxp3 mRNA在肺癌模型组的胸腺表达明显增高(P<0.05),在肺癌模型组脾脏Foxp3 mRNA表达中也略有增加,但增高不显著(P>0.05)。结论:CD4 CD25 调节性T细胞可能在肿瘤免疫耐受中发挥重要作用。     

CD4+CD25+CCR6+调节性T细胞在小鼠乳腺癌模型中对CD8+T细胞的抑制作用

目的：观察CD4+CD25+CCR6+调节性T细胞（简称CCR6+Tregs）体内对CD8+T细胞功能的抑制作用,并探讨其与肿瘤免疫逃逸的关系。方法：建立4T1乳腺癌细胞荷瘤裸鼠模型,FACS分选CCR6+Tregs,检测其Foxp3的表达;FACS分选4T1特异性CD8+T细胞,CFSE标记后分别与CCR6+Tregs或CCR6Tregs共同过继转输入4T1荷瘤裸鼠体内,观察荷瘤裸鼠肿瘤生长情况和小鼠存活时间;FACS检测肿瘤组织中CD8+T细胞的增殖、细胞因子IFNγ的产生和颗粒酶B的表达情况。结果：CCR6+Tregs和CCR6Tregs均高表达Foxp3;CCR6+Tregs和CD8+T细胞共转输组4T1荷瘤裸鼠肿瘤的生长明显快于CCR6Tregs共转输组和CD8+T细胞单转输组,同时该组荷瘤裸鼠生存时间也明显缩短（P<0.05）;CCR6+Tregs和CD8+T细胞共转输组CD8+T细胞的增殖、IFNγ的产生和颗粒酶B的表达均明显低于CCR6Tregs共转输组和CD8+T细胞单转输组（P<0.05）。结论：CCR6+Tregs在体内可以有效抑制CD8+T细胞的功能,其在肿瘤免疫逃逸和肿瘤发生、发展中发挥重要作用。     

鼻咽癌组织与外周血CD4+CD25+调节性T细胞检测及临床意义

目的 通过检测鼻咽癌患者肿瘤组织及外周血中CD4+T、CD8+T、CD4+CD25T、CD4+CD25+T细胞的频数,寻找客观、全面评价鼻咽癌患者免疫状态的临床指标.方法 采用流式细胞术检测40例初诊鼻咽癌患者及10例正常时照鼻咽部组织和外周血CD4+T、CD8+T、CD4+CD25-T、CD4+CD25+T细胞比例.结果 鼻咽癌患者CD4+T细胞比例及CD4+/CD8+T比值均低于对照组(P<0.05),而CD8+T细胞两组间差异无统计学意义(P>0.05),但是CD4+/CD8+T比值在鼻咽癌组织与外周血间差异无统计学意义(P>0.05).鼻咽癌组织及外周血中CD4+CD25+T细胞比例都高于对照组(P<0.05),同时癌组织中该细胞比例远远高于外周血(P<0.05).在鼻咽癌组织中CD4+CD25+T细胞与CD8+T细胞、CD4+CDQ5-T细胞呈负相关(r分别为-0.70、-0.675,P<0.05),而在外周血中没有相关关系(P>0.05).在不同T(原发肿瘤大小)组间,T4组的鼻咽癌组织中CD4+CD25+T细胞分别高于T1、T2、T3各组(P<0.05).而在T1、T2、T3各组间差异无统计学意义(P>0.05);鼻咽癌中CD4+CD25+T细胞比例与患者有无淋巴结转移并无关系(P>0.05);鼻咽癌组织中Ⅲ+Ⅳ期组CD4+CD25+T细胞比例高于Ⅰ+Ⅱ期组(P<0.05),而在外周血中两组间差异无统计学意义(P>0.05).结论 CD4+CD25+T细胞与鼻咽癌病程进展无相关性,但是联合检测患者肿瘤组织及外周血中CD4+CD25+T细胞的频数并结合既往CD4+/CD8+T比值会全面反应患者免疫状态,为临床治疗提供依据.     

水飞蓟素通过调控骨髓来源的抑制性细胞抗肺癌机制的实验研究

目的:研究水飞蓟素通过调控骨髓来源的抑制性细胞（MDSC）的抗肺癌机制。方法:我们在C57/BL6小鼠上构建了路易斯（Lewis）肺癌细胞转移瘤模型，灌胃给予不同浓度(25、50 mg/kg,每天一次)的水飞蓟素。测量小鼠肿瘤体积及体重变化。用免疫组化方法检测Lewis肺癌细胞肿瘤增殖相关指标增殖细胞核抗原（PCNA）的表达。通过TUNEL染色检测肺癌细胞的凋亡情况。通过免疫组化检测组织中CD8+ T细胞浸润及功能。通过流式细胞术检测CD11b+Gr-1+的MDSC百分比。通过Q-PCR检测肿瘤组织中Arg-1、iNOS2及MMP9的表达。结果:25和50 mg/kg的水飞蓟素剂量依赖地抑制肿瘤生长，诱导肿瘤细胞凋亡。水飞蓟素增加了CD8+ T细胞浸润；减少了肿瘤组织中MDSC的比例，肿瘤组织中Arg-1、iNOS2 及MMP9（MDSC功能相关） mRNA表达的减少也证实了MDSC功能的减弱。结论:水飞蓟素抑制了MDSC，促进CD8+ T细胞浸润及功能，抑制模型中肿瘤生长，为水飞蓟素治疗肺癌提供了依据。     

恶性肿瘤患者外周血CD4+CD25+调节T细胞的检测及其临床意义

目的探讨恶性肿瘤外周血CD4+CD25+调节T细胞水平的特点及其临床意义.方法采用流式细胞术检测53例恶性肿瘤患者外周血CD4+CD25+调节T细胞水平及淋巴细胞亚群,并进行分层分析.结果外周血淋巴细胞亚群分析显示恶性肿瘤患者CD4、CD16阳性细胞比例在早期(Ⅰ+Ⅱ期)患者即低于对照组,但差异无显著性意义(P＞0.05),CD4/CD8亦低于对照组,差异有显著性意义(P＜0.05);随着疾病进展(Ⅲ、Ⅳ期)CD4、CD4/CD8、CD16阳性细胞比例减低明显,差异有显著性意义(P＜0.05);各期CD8、CD19、CD3与对照组接近(P＞0.05).外周血CD4+CD25+调节T细胞比例健康对照组CD4+CD25+T细胞水平为(14.49±4.69)%,恶性肿瘤53例CD4+CD25+T细胞比例为(19.61±8.17)%,统计学有差异(P＜0.05);进一步分层分析显示随疾病进展外周血CD4+CD25+T细胞水平升高,在肿瘤进展期(Ⅳ期)尤其明显,统计学有极显著差异(P＜0.01).结论恶性肿瘤患者外周血CD4+CD25+调节T细胞水平的升高,与恶性肿瘤免疫功能低下及肿瘤的发生发展密切相关.去除这群细胞可有效诱导肿瘤免疫,为肿瘤治疗提供一种新的方法.     

Enhanced myeloid specificity of CD117 compared with CD13 and CD33

The c-kit proto-oncogene encodes a 145 kd tyrosine kinase transmembrane receptor, which plays a key role in haemopoiesis. The c-kit has been classified as CD117 and is especially useful in the differential diagnosis of acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL). We analysed 104 consecutive cases (55 AML, 23 B-cell lineage ALL, three T-cell ALL, 11 blast crisis of chronic myeloproliferative disorders and 12 cases of myelodysplastic syndromes with more than 10% of blasts) referred to our Hospital for immunophenotypic diagnosis and compared the expression pattern of CD13, CD33 and CD117 using the same fluorochrome (phycoerythrin-PE). The recommendations of the EGIL group were followed in order to establish lineage involvement of the blastic population. The threshold used to assign positivity for CD117 was 10%. Bcr/abl, TEL/AML-1 and MLL rearrangements were assessed by molecular methods. CD117 expression was detected in 91% of AML and MDS. All the negative cases corresponded to acute monocytic leukemias. The calculated specificity for myeloid involvement was 0.86 for CD117, 0.36 for CD13 and 0.44 for CD33 (P < 0.005). CD117 was also positive in four cases of ALL. None of these cases showed bcr/abl or MLL rearrangements. In the light of these findings, CD117 expression should yield a higher score, at least one point, in the system currently applied for the diagnosis of biphenotypic acute leukemias (BAL) as its myeloid specificity is greater than that of CD13 and CD33. Moreover, its absence in AML could identify two subgroups of M5b cases. The coexpression of CD117 with cytoplasmic CD79a is often associated with CD7 reactivity, suggesting a stem cell disorder. CD117 should be included on a routine basis for the immunophenotypic diagnosis of acute leukemias.     

CD5, CD10, and CD23 expression in Waldenstrom's macroglobulinemia

CD5, CD10, and CD23 are cell surface antigens used to distinguish B-cell disorders. The expression of these antigens and their clinical significance in Waldenstrom's macroglobulinemia (WM), an uncommon B-cell disorder, remains to be clarified. We therefore determined expression of CD5, CD10, and CD23 by flow cytometric analysis on bone marrow lymphoplasmacytic cells (CD19+ k/l light chain restricted) for 171 serially biopsied patients with findings of the consensus panel definition of WM. Importantly, we also correlated laboratory and clinical data, as well as existence of a familial history of a B-cell disorder in view of reports suggesting familial predisposition in WM. These studies demonstrated tumor cell expression of CD5, CD10, and CD23 in 15 of 171 patients (9%), 11 of 161 patients (7%), and 37 of 105 patients (35%), respectively. Coexpression of CD23 with CD5 or CD10 was common. Tumor Lymphoplasmacytic from 10 of 15 (66%) and 3 of 11 (27%) patients with WM that expressed CD5 and CD10, respectively, also showed expression of CD23 (P = 0.01 and P = 0.08, respectively). Among patients with CD23 expression, increased serum immunoglobulin (Ig) M levels were observed compared with patients without CD23 expression (P = 0.05). No differences in age at diagnosis; presence of adenopathy and/or splenomegaly; bone marrow involvement; serum IgA, IgB, and b2 macroglobulin levels; hematocrit; platelet count; or familial history of WM or a related B-cell disorder were observed among patients with and without CD5, CD10, and CD23 expression. These studies demonstrate that CD5, CD10, and CD23 are commonly found in WM and that their expression should not exclude the diagnosis of WM. Moreover, expression of CD23 may define a clinically distinct subset of patients with WM.     

人CD46、CD55和CD59与肿瘤免疫逃逸及免疫治疗

膜结合补体调节蛋白CD46、CD55和CD59在肿瘤细胞膜上表达或过表达,保护肿瘤细胞免受免疫系统的攻击,成为肿瘤细胞免疫逃逸的途径之一.如何下调肿瘤细胞表面三者表达或抑制其功能以增强其对补体依赖的细胞毒作用的敏感性备受关注.     

CD46 CD55 CD59在结肠癌组织中的表达及意义

目的:检测CD46、CD55、CD59在结肠癌组织中的表达情况,分析其与结肠癌临床病理参数间的相关性及意义。方法:选取有详细性别、年龄、组织分化、病理分期、肿瘤部位、组织类型资料的组织芯片标本,包括121 例结肠癌和121 例癌旁结肠组织,均为2004年10月至2006年6 月第四军医大学西京消化病医院胃肠外科手术切除标本。应用免疫组织化学改良二步法分别检测CD46、CD55、CD59的表达情况。结果:CD46、CD55及CD59在结肠癌组织中的阳性表达率均显著高于对应的癌旁组织（P < 0.001）。 CD46表达水平与性别、年龄、组织分化、TNM 病理分期、肿瘤位置、病理组织类型均无关（P > 0.05）。 CD55、CD59的表达水平与性别、年龄、肿瘤位置、病理类型无关（P > 0.05）,而与组织分化、TNM 病理分期有关（P < 0.05）。 其表达强度阳性率中低分化组明显高于高分化组（P < 0.05）。 TNM 分期中Ⅲ、Ⅳ期患者肿瘤病理组织强阳性表达率高于Ⅰ、Ⅱ期阳性表达率,两者比较有统计学意义（P < 0.05）。 结论:CD46、CD55及CD59在结肠癌组织中高表达,特别是CD55、CD59的表达与肿瘤分化、病理分期相关,提示三者表达水平与结肠癌生物学行为密切相关。      

Poor prognosis of mediastinal non-Hodgkin's lymphoma with an immature phenotype of CD2+, CD7 (or CD5)+, CD3-, CD4-, and CD8-

Nine children with mediastinal non-Hodgkin's lymphoma (NHL) were treated according to our new regimen which is characterized by intensified therapy with high-dose cytosine arabinoside (HDCA). After induction therapy with a combination of five drugs, such as vincristine, doxorubicin, cyclophosphamide, 1-asparaginase, and prednisolone, intermediate dosages of methotrexate (MTX) (1 g/m2) and HDCA (1.5 g/m2 x 12 doses) were administered. All but one patient (88.9%) achieved complete remission and then received this intensified therapy. With a median follow-up period of 25.5 months, five patients are still in complete remission, but three patients have relapsed. From the phenotypic point of view, these relapsed patients showed only very immature T-cell differentiation antigens such as CD2 and CD7 (or CD5). These results suggest that HDCA as intensified therapy for children with mediastinal NHL seems to be effective. However, for patients with an immature phenotype of T-lineage cells, more sophisticated regimens should be prepared.     

Agranular CD4+/CD56+ cutaneous neoplasm

CD4+ CD56+ cutaneous neoplasm with hematological relapse is a rare malignant disease and has been described recently in the literature as blastic or agranular NK-cell leukemia/lymphoma. The origin of this neoplasm is uncertain. We describe a 75-year-old patient with a primary cutaneous neoplasm CD4+ CD56+ who evolved to leukemic phase despite standard lymphoma chemotherapy. Morphologically, the cells were undifferentiated without granules in the cytoplasm. The immunophenotype showed the expression of CD4, CD56, CD68, CD33, CD7, CD2, CD45RA, and CD38. Histological analysis revealed a cell infiltration mainly located in the dermis. T-cell receptor and immunoglobulin heavy chain genes were in germline configuration. Cytogenetic study showed complex structural abnormalities with a deletion of the chromosome 5 del(5q). The clinical course was aggressive with an early hematological relapse.     

The CD11/CD18 leukocyte glycoprotein deficiency

CD11/CD18 leukocyte glycoprotein deficiency is a rare, inherited disorder of leukocyte function, manifested by recurrent severe bacterial infections. A deficiency in the expression of a family of leukocyte membrane glycoproteins (the CD11/CD18 glycoproteins) represents the molecular basis for this disease.