乳腺癌患者围手术期NK细胞及其亚群的动态变化

目的探讨乳腺癌患者围手术期NK细胞及其亚群的变化及其临床意义。方法以乳腺良性肿瘤患者8例、健康人14例为对照,乳腺癌患者29例手术前后,分别用流式细胞术测定外周血NK细胞及其亚群的变化。结果乳腺癌患者外周血淋巴细胞群中CD56＋细胞和CD3-CD16＋CD56＋的百分率以及CD56＋细胞群中CD3-CD16＋CD56dim的百分率等3个指标均较健康对照和良性乳腺良性肿瘤组明显低下（P值均为0.000）,CD56＋细胞群中CD3-CD16＋CD56bright细胞的百分率与两对照组比较差异无显著性。乳腺癌患者术后1 d上述3指标均较术前明显下降（P值均为0.000）,术后7 d,外周血淋巴细胞群中CD56＋细胞的百分率仍明显低于术前（P=0.024）,另两指标与术前差异无显著性,术后1 d和7 d外周血CD56＋细胞群中CD3-CD16＋CD56bright细胞的百分率与术前的差异均无显著性。结论乳腺癌患者NK细胞及其亚群CD3-CD16＋CD56dim明显低于健康对照和乳腺良性肿瘤患者,手术应激会导致其进一步下降,围手术期应密切观察NK细胞尤其是CD3-CD16＋CD56dim亚群的变化,并给予适当的免疫干预。     

人外周血CD56+NK细胞亚群表型和生物学特征

目的 探讨人CD56^＋NK细胞亚群、表型和生物学特征。方法 分离正常人外周血单个核细胞,利用多种抗体进行细胞表面和细胞内细胞毒效应分子（颗粒酶B和穿孔素）染色,通过流式细胞仪,在单个细胞水平上分析CD56^＋NK细胞亚群的表型及其生物学特征。结果 根据CD56分子表达密度的不同,将人外周血中NK细胞分为CD56^bright出和CD56^dim两大亚群。CD56^bright和CD56^dim细胞均表达CD95,CD56^bright细胞亚群中,CD95^bright和CD95^dim表达百分率分别为21．4％和77．0％;CD56^dim细胞亚群中,CD95^bright和CD95^dim表达百分率分别为23．1％和75．6％。与CD56^bright出细胞亚群相比,CD56^bright细胞表达CD8、颗粒酶B和穿孔素细胞的阳性率较高,分别为9．1％、6．2％和8．O％。在CD56^bright和CD56^bright亚群中,CD95^brightCD8^＋亚群高表达颗粒酶B和穿孔素。结论 人外周血CD56^＋NK细胞由不同表型和生物学特征的细胞亚群组成,CD56^dim”和CD56^dimCD8^＋NK细胞亚群在杀伤破坏靶细胞中发挥重要作用。     

CD8+CD28-Ts、CD3+CD56+NKT细胞在B细胞非霍奇金淋巴瘤患者外周血中分布的分析

背景及目的:目前认为B细胞非霍奇金淋巴瘤(B-NHL)常伴随免疫抑制,CD8 CD28-Ts(Ts)细胞和CD3 CD56 NKT(NKT)是新鉴定的新型免疫抑制性调节细胞,在肿瘤的免疫抑制及免疫逃逸机制中起重要作用,但它们在B细胞淋巴瘤患者外周血的分布情况及其免疫抑制中的作用目前尚不清楚。本文通过分析两者在化疗前及化疗后B-NHL患者外周血中比例及变化规律,初步探讨它们在B-NHL的免疫抑制作用及其影响因素,为有效干预患者的免疫功能提供参考。方法:应用流式细胞仪检测79例治疗前的B-NHL患者、经4～6周期化疗后完全缓解(CR)的18例患者、30例健康志愿者外周静脉血中NKT及Ts细胞的比例。结果:在79例治疗前B-NHL患者的外周血中,Ts细胞比例为(18.19±5.03)%,较正常对照组(11.20±3.49)%明显增高(P<0.01);NKT的比例为(6.08±3.29)%,亦较正常对照组的(3.52±1.56)%明显增高(P<0.01)。Ts在不同临床分期的患者之间无显著性差异P>0.05;Ⅰ期为(17.56±4.10)%、Ⅱ期为(18.05±5.64)%、Ⅲ期为(18.14±5.58)%、Ⅳ期为(18.95±4.64)%;在不同恶性程度的患者之间亦无显著性差异P>0.05;低度恶性为(17.81±5.24)%、中度恶性为(18.37±4.83)%、高度恶性为(18.31±5.93)%;在治疗前(18.64±4.55)%和CR后(19.42±4.95)%患者之间亦无显著性差异(P=0     

晚期非小细胞肺癌患者外周血CD4~+ CD25~+ FOXP3~+调节性T细胞的表达及临床意义

目的探讨晚期非小细胞肺癌患者外周血CD4~+ CD25~+ FOXP3~+ 调节性T(Treg)细胞的表达及其临床意义。方法采用免疫荧光术及流式细胞仪检测50例晚期非小细胞肺癌患者及50例健康对照组外周血中CD4~+ CD25~+ FOXP3~+ Treg细胞、CD4~+ CD25~+ Treg细胞、CD4~+ T细胞和CD4~+ CTLA-4~+ T细胞的表达。结果晚期非小细胞肺癌患者外周血中CD4~+ CD25~+ FOXP3~+ Treg细胞、CD4~+ CD25~+ Treg细胞和CD4~+ CTLA-4~+ T细胞的比例均高于健康对照组(均P<0.05),CD4~+ T细胞的比例均低于健康对照组(均P<0.05)。结论晚期非小细胞肺癌患者外周血CD4~+ CD25~+ FOXP3~+ Treg细胞比例高于健康对照者,可能与肺癌患者的免疫抑制和肿瘤进展相关。     

乳腺癌患者外周血CD4+CD25+调节性T细胞的检测及意义

目的探讨乳腺癌患者外周血中CD4+CD25+调节性T细胞的变化及意义.方法采用流式细胞技术检测64例乳腺癌患者外周血中CD4+CD25+调节性T细胞,采用ELISA法检测外周血中转化生长因子-β1(TGF-β1)的表达水平.结果乳腺癌患者外周血中CD4+CD25+调节性T细胞占T淋巴细胞的百分比为(5.1±2.9)%,高于乳腺良性肿物患者和健康志愿者(P均<0.05).乳腺癌患者外周血中CD4+CD25+T细胞水平与肿物大小、TGF-β1呈正相关(r分别为0.511和0.253),与CD8+CD28+T细胞和NK细胞呈负相关(r分别为-0.243和-0.301).结论乳腺癌患者外周血中具有免疫抑制活性的CD4+CD25+调节性T细胞水平较高,对乳腺癌患者具有免疫抑制作用.     

CD8+CD25+FOXP3+调节性T淋巴细胞在恶性胸腔积液中的表达及其临床意义

目的 通过检测恶性胸腔积液中CD8+CD25+Foxp3+调节性T淋巴细胞(T8reg)的表达,探讨其与恶性胸腔积液患者临床预后的关系.方法 同步采集30例肺癌合并胸腔积液患者的胸腔积液和外周血,20例良性胸腔积液患者的胸腔积液和外周血,另采集20例健康对照者外周血,用流式细胞术检测上述标本中CD8+CD25+Foxp3+T淋巴细胞的表型、百分比,分析其与恶性胸腔积液患者生存时间的关系.结果 恶性胸腔积液组中T8reg占总CD8+T细胞的比例显著高于良性胸腔积液组[(2.20±0.25)%vs(0.38±0.05)%,P=0.018],亦高于自身外周血组[(0.52±0.06)%,P=0.000],恶性胸腔积液患者外周血中T8reg的比例高于正常健康者外周血中的比例[(0.52±0.06)%vs(0.31±0.04)%,P=0.005].而良性胸腔积液组胸腔积液、外周血(0.34±0.04)%与健康对照组外周血三组中T8reg细胞的数量占总CD8+T细胞比例没有明显升高,差别没有统计学意义(P＞0.05).T8reg高、低表达水平组患者的中位生存时间分别为105、195d,两者差异有统计学意义(P=0.004).Cox回归模型多因素分析显示MPE中T8reg的表达水平、肿瘤大小是影响MPE患者预后的独立因素(P值分别为0.018、0.006).结论 肺癌伴胸膜转移患者的MPE及其外周血T8reg细胞的比例明显增高;MPE中T8reg细胞表达下降预示MPE患者生存率会明显改善,提示CD8+CD25+Foxp3+T细胞在肺癌发生、发展的免疫病理过程中具有显著意义.     

人外周血细胞毒性CD3-CD56+ NK细胞高效扩增的研究

 目的　探索从人PBMC中高效扩增细胞毒性CD3-CD5 6 + NK细胞的方法。方法　使用干细胞生长培养基 (SCGM )和RPMI 16 4 0培养基 ,在抗CD3单抗、IL 2和植物血凝素 (PHA)的作用下从 5例健康成人PBMC中诱导扩增CD3-CD5 6 + NK细胞 ,并用MTT法检测其细胞毒活性。结果　只有在使用SCGM为基础培养基时 ,PBMC经抗CD3单抗、IL 2作用获得大量增殖 ,在PHA存在时获得最大增殖(P <0 .0 5 ) ,在 14天时扩增 5 1.3± 7.2倍 ,含有 (5 2 .4± 7.9) %CD3-CD5 6 + NK细胞和 (14 .2± 4 .0 ) %CD3+ CD5 6 + T细胞 ;在效 /靶比 10∶1时 ,对K5 6 2和Raji的杀伤率分别达83.7%和 5 5 .8%。结论　可使用SCGM ,在抗CD3单抗、IL 2和PHA协同作用下大量扩增细胞毒性CD3-CD5 6 + NK细胞 ,为应用NK细胞进行肿瘤过继免疫治疗提供了一种简单有效的扩增NK细胞的方法。     

急性白血病伴CD56阳性的临床意义

目的：探讨CD56在急性白血病中的表达及其临床意义。方法：就近2年来应用流式细胞仪检测了CD56的92例急性白血患者中发现的CD56阳性病例,分析细胞形态学、免疫表型和临床特点。结果：92例急性白血病中15例（16．3％）表达CD56,其中1例急性前髓系／NK细胞白血病（Myeloid/NK cell precursor acute leukemia),1例原始NK细胞白血病（Blastic NK cell leukemia),1例NK样T细胞淋巴瘤／白血病（NK－like T-cell lymphoma/leukemia),12例急性髓细胞白血病伴NK细胞抗原表达或急性髓系／NK细胞白血病。结论：伴CD56阳性急性白血病,其细胞形态学、免疫表型及临床表现各有特点,见于M2、M5、L2,髓外浸润多见,多预后不良。     

肺癌患者外周血 T- 淋巴细胞亚群 NK 细胞的基线数值及其与预后的关系*

目的:对比肺癌患者与健康者之间淋巴细胞亚群差异,评估肺癌患者外周血T 淋巴细胞亚群及自然杀伤细胞（NK）之基线值与预后的关系。方法:收集2006年2 月至2013年3 月就诊于天津医科大学肿瘤医院病例资料完整的肺癌患者105 例,其中非小细胞肺癌（NSCLC ）86例、小细胞肺癌（SCLC）19例,另选健康对照35例,对比接受治疗前肺癌患者和健康对照者外周血中的CD3+T 细胞、CD4+T 细胞、CD8+T 细胞及NK细胞所占百分比,并回顾性分析86例NSCLC 患者初治时外周血淋巴细胞亚群与预后的关系。结果:肺癌患者外周血CD3+T 细胞、CD4+T 细胞、NK细胞及CD4/CD 8 比值均明显低于健康对照组（P = 0.011,P = 0.007,P <0.001,P=0.025）,而CD8+T 细胞比例高于健康对照组（P = 0.013）。 当CD8+T ≥ 31.8% 及CD4/CD 8< 1.28时NSCLC 患者可以获得一个更长的OS（中位OS分别为36.2 m vs . 20.0 m ,P = 0.010;30.8 m vs . 20.0 m ,P = 0.035）。 而CD3+T 细胞、CD4+T 细胞及NK细胞百分比对NSCLC 患者预后无显著影响。结论:外周血CD8+T 细胞基线水平较高的NSCLC 患者生存较长,此基线水平可能对NSCLC 患者预后有指示作用。      

CD4+CD25+Tr细胞与非小细胞肺癌相关性研究

目的：探讨CD4^＋CD25^＋Tr细胞与非小细胞肺癌的相关性。方法：采用流式细胞技术分别检测60例非小细胞肺癌患者和20例健康者外周血中CD4^＋CD25^＋Tr细胞数量。结果：非小细胞肺癌患者外周血中CD4^＋CD25^＋Tr细胞表达水平明显高于健康者（P〈0.05）,且与临床分期呈正相关。结论：非小细胞肺癌患者外周血中CD4^＋CD25^＋Tr细胞数量的增多与非小细胞肺癌发病有关。     

Ex vivo expansion of CD8+CD56+ and CD8+CD56- natural killer T cells specific for MUC1 mucin

Prostate cancers express MUC1, but nearly all metastatic cells lack HLA class I molecules. Thus, a lymphocyte population that can sense its antigenic environment, while also able to react to stimuli of natural killer (NK) cells, may be a more versatile effector cell population for antitumor immune responses. Herein, we report that tumor-specific MUC1 peptide, interleukin 2, and interleukin 12 act synergistically to stimulate the ex vivo expansion of CD8(+)CD56(-) T cells and CD8(+)CD56(+) natural killer T (NKT) cells from the peripheral blood mononuclear cells of prostate cancer patients, as well as healthy male and female donors. Both the CD56(+) NKT cells and CD56(-) T cells lysed allogeneic mucin-bearing target cells, as well as NK target cells, but not lymphokine-activated killer target cells. However, the CD56(+) NKT cells displayed a 2-fold greater cytolytic activity than the CD56(-) T cells. The mucin-specific cytolytic activity and NK cytolytic activities for both lymphocyte populations were independent of HLA class I and CD1 molecules. The CD56(-) T cells up-regulated CD56 with continued antigenic stimulation in the presence of interleukin 12, suggesting that CD8(+)CD56(-) T cells are NKT cells. However, CD56(+) NKT cells expand poorly to continued stimulation. All mucin-stimulated NKT cells exhibited the activated/memory CD45RO phenotype. The NKT cell lines express the alpha/beta T-cell receptor (TCR). The TCR repertoire was limited and varied with cell line, but was not the V alpha 24V beta 11 TCR typically associated with NKT cells. Whereas CD161 is generally considered a marker of NKT cells, the mucin-stimulated NKT cells did not express this marker. Thus, we have described two phenotypically distinct NKT types that do not display a biased TCR repertoire, but do display specificity for a tumor-specific peptide antigen (CTL-like activity), as well as HLA class I-deficient target cells (NK-like activity).     

CD3+CD56+CD8+ cells demonstrating a suppressor T cell-like function in the peripheral blood of colon cancer patients

We previously reported that HLA-unrestricted CTLs against MUC-1 were induced from colon cancer patients by stimulating peripheral blood lymphocytes (PBLs) with recombinant MUC-1 vaccinia virus (rVMUC-1). We have performed adoptive immunotherapy (AI) for two gastric and two colon cancer patients, using the rVMUC-1-stimulated T lymphocytes. A significant level of HLA-unrestricted cytotoxicity against MUC-1 was induced in the two colon cancer patients (pA and pB) during the first adoptive immunotherapy, but extremely reduced during the second AI. During the second stimulation phase, the rate of CD3+CD56+CD8+ cells were significantly increased and that of CD3+CD56-CD4+ cells were significantly decreased in the two colon cancer patients as compared to the first AI. CD3+CD56+CD8+ and CD3+CD56-CD4+ cells were isolated from the second AI of the colon cancer patient (pB) and designated as D856 and D4, respectively. The D4 cells demonstrated a high level of HLA-unrestricted CTL activity against MUC-1, but D856 cells did not. When D856 cells were mixed with D4 cells at a D856/D4 ratio of 1:3, 1:2, and 1:1 and used as effector cells, the HLA-unrestricted and MUC-1-specific CTL activity of D4 cells was suppressed in a D856/D4 ratio-dependent manner. Further, D856 cells were highly lytic for the D4 cells demonstrating HLA-unrestricted cytotoxicity against MUC-1. It is concluded that the reduction in HLA-unrestricted cytotoxicity against MUC-1 during the second AI is attributed to the D856 cells killing MUC-1-specific CTLs (D4). Thus, the CD3+CD56+ CD8+ cells seem likely to behave as a suppressor T cell.     

Tim-3 Expression by Peripheral Natural Killer Cells and Natural Killer T Cells Increases in Patients with Lung Cancer - Reduction after Surgical Resection

Background: The purpose of this study was to investigate Tim-3 expression on peripheral CD3-CD56+natural killer (NK) cells and CD3+CD56+ natural killer T (NKT) cells in lung cancer patients. Materials andMethods: We analyzed Tim-3+CD3-CD56+ cells, Tim-3+CD3-CD56dim cells, Tim-3+CD3-CD56bright cells, and Tim-3+CD3+CD56+ cells in fresh peripheral blood from 79 lung cancer cases preoperatively and 53 healthy controlsby flow cytometry. Postoperative blood samples were also analyzed from 21 members of the lung cancer patientcohort. Results: It was showed that expression of Tim-3 was significantly increased on CD3-CD56+ cells, CD3-CD56dim cells and CD3+CD56+ cells in lung cancer patients as compared to healthy controls (p=0.03, p=0.03 andp=0.04, respectively). When analyzing Tim-3 expression with cancer progression, results revealed more elevatedTim-3 expression in CD3-CD56+ cells, CD3-CD56dim cells and CD3+CD56+ cells in cases with advanced stages(III/IV) than those with stage I and II (p=0.02, p=0.04 and p=0.01, respectively). In addition, Tim-3 expression wassignificantly reduced on after surgical resection of the primary tumor (p<0.01). Conclusions: Tim-3 expressionin natural killer cells from fresh peripheral blood may provide a useful indicator of disease progression of lungcancer. Furthermore, it was indicated that Tim-3 might be as a therapeutic target.     

Numbers and cytotoxicities of CD3+CD56+ T lymphocytes in peripheral blood of patients with acute myeloid leukemia and acute lymphocytic leukemia

Recent reports have highlighted the role of cellular immunity in anti-tumor defenses. T lymphocytes are known to play important part in anti-cancer immunity. The number and function of T lymphocytes are altered in chronic leukemia patients. CD3⁺CD56⁺ T lymphocytes have also been found to be abnormal in cancer patients. We therefore investigated changes in the number and cytotoxicity of CD3⁺CD56⁺ T lymphocytes in the peripheral blood of acute leukemia (AL) patients (excluding acute promyelocytic leukemia), to improve our understanding of the role of this T lymphocyte subset. We analyzed CD3⁺CD56⁺ T lymphocyte numbers and cytotoxicities in healthy controls, AL patients, and AL patients with complete remission. Lymphocyte counts were performed in peripheral blood and flow cytometry was used to determine cell numbers and cytotoxicities. The absolute number of CD3⁺CD56⁺ T lymphocytes was increased in AL patients (including acute myeloid [AML] and acute lymphocytic leukemia [ALL]) compared with healthy controls (P < 0.05), but their functioning was significantly reduced (P < 0.05). The number of CD3⁺CD56⁺ T lymphocytes in AML and ALL patients who achieved remission following chemotherapy was close to healthy controls (P > 0.05), but their functioning was still significantly reduced (P < 0.05). In addition, the number of CD3⁺CD56⁺ T lymphocytes increased significantly in AML patients with increased peripheral blood white blood cell (WBC) counts, and in ALL patients without increased WBCs. These results suggest that cellular immunity may respond to AML and ALL, but that lymphocyte cytotoxicity remains impaired. Dysfunction of CD3⁺CD56⁺ T lymphocytes in AML and ALL patients may contribute to the failure of the host immune response against leukemic blasts.     

CD1a and CD1d Genes Polymorphisms in Breast,Colorectal and Lung Cancers

CD1 molecules might contribute to anti-tumor immune response by presentation of tumor-derived lipid and glycolipid antigens to T cells and NKT cells. Polymorphisms in CD1 genes have been suggested to modify ligand binding of CD1 molecules and thereby change the antigen presenting ability of these molecules. The aim of this study was to investigate the exon 2 polymorphisms of CD1a and CD1d in several high incident cancers in Iran. For this purpose, 201 female breast cancer patients and 207 healthy women, 64 lung cancer patients and 95 healthy individuals and 109 patients with colorectal cancer and 109 healthy controls were recruited to this study. Using PCR-SSP method, no significant correlation was found in genotype and allele frequencies of CD1a between all three studied groups and their control counterparts. Moreover, a dominant frequency of CD1d 01 (A) allele was observed in the majority of studied individuals. No significant association between the CD1 polymorphisms and prognostic factors in breast, lung and colorectal cancers was detected. Our results highlight the conserved nature of CD1 genes and may point to the immuoregulatory functions of CD1 molecules in cancer that can be exerted through fine tuning of NK, T and NKT cells.     

肺癌患者外周血免疫细胞毒细胞的检测

 目的 探讨肺癌患者群体的免疫细胞毒细胞的功能状态。方法 应用流式细胞仪测定30例手术前肺癌患者和30例该院健康职工外周血总T淋巴细胞、自然杀伤细胞（NK）、NK样T杀伤细胞（NKT）、γδT细胞和CTL细胞。结果 γδT细胞：正常组为6.54±2.94，患者组为3.58±2.14，t = 3.730，P =0.001。CTL细胞：正常组为17.21±4.49，患者组为10.86±4.67，t =5.130，P＜0.001；其他被测细胞的百分率，经统计学分析，均无统计学意义。结论 特异性免疫杀伤细胞低下对肺癌的发生有重要影响。非特异性免疫细胞毒细胞影响有限。     

The phenotype of ex vivo generated cytokine-induced killer cells is associated with overall survival in patients with cancer

Cytokine-induced killer (CIK) cells are ex vivo generated heterogeneous NK-like T lymphocytes. It is not very clear whether the phenotype of CIK cells is associated with their therapeutic efficacy to cancer patients. Thus, in this study, the association of phenotype of CIK cells and the overall survival of 121 patients with hepatocellular carcinoma (HCC), 74 patients with lung cancer and 42 patients with colorectal cancer, all of whom underwent surgical resection and received autogenous CIK cell therapy, was analyzed. We found that high ratio of the CD3+CD4+ subset was associated with poorer overall survival in colorectal cancer, but not HCC or lung cancer. A high ratio of the CD3+CD8+ subset was associated with improved overall survival in all three types of cancer. A high ratio of the CD3+CD56+ NK-like subset was associated with improved overall survival in lung and colorectal cancer, but not HCC. A high ratio of the CD3-CD56+ NK subset was associated with poorer overall survival in lung and colorectal cancer, but not HCC. In conclusion, the CD3+CD8+ and CD3+CD56+ subsets, especially the CD3+CD8+ subset, may be the major phenotypes responsible for anti-tumor immunity in vivo after autogenous CIK cell therapy.     

CD4+ T lymphocytes: a critical component of antitumor immunity

Both prophylactic and therapeutic vaccines targeting a wide variety of cancers are being developed. Because of the potency of cell-mediated immunity, many vaccine strategies are focused on activating tumor-specific cytotoxic CD8⁺ T lymphocytes. CD4⁺ T lymphocytes are a key element in optimal activation of CD8⁺ T cells and in the maintenance of immune memory, and therefore their activation is critical for cancer vaccine efficacy. This article reviews the mechanisms by which CD4⁺ T cells facilitate tumor immunity and the vaccine strategies that enhance CD4⁺ T cell activity.