Phenotypic and functional analysis of tumor-infiltrating lymphocytes compared with tumor-associated lymphocytes from ascitic fluid and peripheral blood lymphocytes in patients with advanced ovarian cancer.

To investigate and compare the phenotype and function of lymphocytes collected from patients harboring advanced ovarian cancer, leukocytes from peripheral blood (n = 18), ascitic fluid (n = 13) and tumor tissues (n = 13) were evaluated for the relative proportions of lymphocyte subsets, including CD3+, CD4+, CD8+, CD19+, CD56 and the early (CD25) and late (HLA-DR) activation markers on CD3+ T cells. The ability to synthesize type 1 cytokines (IFN-gamma and IL-2) and a type 2 cytokine (IL-4) was assessed by flow cytometry. In all patients, T cells (CD3+) were the major leukocyte population detected in each tissue, with CD4+ T cells being dominant in peripheral blood lymphocytes (PBL) and tumor-associated lymphocytes (TAL) but not in tumor-infiltrating lymphocytes (TIL) (CD4:CD8 ratios: 3.0 vs. 2.0 vs. 1.0, respectively). CD19+ lymphocytes (B cells) and CD56+ lymphocytes (NK cells) were significantly higher in PBL compared to TAL and TIL (p < 0.05). TAL and TIL had a higher proportion of T cells expressing the late activation marker HLA-DR compared to PBL. In contrast, no significant differences were detected in PBL, TAL and TIL in the expression of the early activation marker CD25. Type 1 cytokines were the dominant type produced by in vitro stimulated T cells for each population, with a greater proportion of IFN-gamma+ T cells in TAL and TIL compared to PBL (p < 0.01), and a higher proportion of IL-2+ T cells in PBL compared with TAL and TIL (p < 0.05). Low percentages of IL-4+ T cells (i.e. Th2) were detected in each tissue. Taken together, these data demonstrate the recruitment and accumulation of high concentrations of antigen-experienced T lymphocytes in TAL and TIL compared to PBL. However, low surface expression of IL-2 receptor (i.e. CD25), as well as depressed intracellular IL-2 production in chronically stimulated TAL and TIL suggests that the impaired antitumor function commonly detected in these lymphocyte populations may be secondary to an acquired dysregulation of the IL-2 pathway.     

Induction of tumor-specific cytotoxicity in tumor infiltrating lymphocytes by HPV16 and HPV18 E7-pulsed autologous dendritic cells in patients with cancer of the uterine cervix

OBJECTIVE: To evaluate the potential of autologous dendritic cells (DC) pulsed with HPV16 and HPV18 E7 oncoprotein in restoring tumor-specific cytotoxicity in populations of tumor infiltrating lymphocytes (TIL) for adoptive immunotherapy of cervical cancer patients. METHODS: Full-length E7-pulsed DC-stimulated CD8(+) T cells derived from peripheral blood (PBL) and from tumor tissues (TIL) were tested and compared for their ability to induce a HLA class-I-restricted cytotoxic T lymphocyte (CTL) response against autologous tumor cells. In addition, in order to correlate cytotoxic activity by CTL with a particular lymphoid subset, analysis of surface antigens and intracellular CD3 zeta chain and two-color flow cytometric analysis of intracellular cytokine expression (IFN-gamma vs IL-4) at the single cell level were performed. RESULTS: DC stimulation induced powerful cytotoxicity against autologous tumor target cells by TIL-derived CD8(+) T cells from all three cervical cancer patients, while autologous Epstein-Barr virus-transformed lymphoblastoid cell lines were not lysed. Killing of autologous tumor cells was higher by CD8(+) T cells from TIL compared to PBL (P > 0.01) and was more strongly inhibited by anti-HLA class I MAb (P > 0.05). Phenotypically, all CTL populations were CD3(+)/CD8(+), with higher levels of CD56 expression by TIL-derived CTL. Finally, although a marked Type 1 cytokine bias (i.e., IFN-gamma(high)/IL-4(low)) was observable in both PBL- and TIL-derived DC-stimulated CD8(+) T cell populations, TIL-derived CD8(+) T cells showed a higher percentage of IFN-gamma-positive cells compared to PBL. CONCLUSIONS: Full-length E7-pulsed DC can consistently restore strong CD8(+) CTL responses against autologous HPV16- and HPV18-infected cervical cancer cells. DC-stimulated TIL may represent a superior source of tumor-specific CTL compared to PBL for adoptive T cell immunotherapy of patients harboring metastatic or recurrent cervical cancer refractory to standard treatment modalities.     

葡萄球菌肠毒素激活肿瘤浸润淋巴细胞及外周血淋巴细胞体外抗人卵巢癌的对比研究

目的:探讨葡萄球菌肠毒素A(SEA)对卵巢癌肿瘤浸润淋巴细胞(TIL)及其外周血淋巴细胞(PBL)抗瘤活性的诱导作用。方法:取10例卵巢癌伴腹水患者实体瘤、腹水及外周血标本,分离TIL和PBL。在SEA及IL-2作用下培养,定时计数,了解其增殖情况;流式细胞仪检测其CD3、CD4、CD8表达;噻唑蓝(MTT)比色法测定其对K562及自体肿瘤细胞的细胞毒活性;酶联免疫吸附试验(ELISA)测定培养上清液中TNF-a和IFN-γ浓度。结果:10例中8例成功分离实体瘤TIL、腹水TIL及PBL。(1)SEA刺激的实体瘤TIL、腹水TIL及PBL增殖速率明显较IL-2诱导组快(P<0.05),但增殖高峰后出现下降趋势,IL-2组未出现此现象;(2)CD3+CD4+及CD3+CD8+T表达率均明显上升,其中SEA诱导组比IL-2组增加比例明显(P<0.05),以SEA作用的CD3+CD8+T比例增加最快;(3)TIL对自体肿瘤细胞的杀伤活性明显高于对K562细胞的杀伤活性(P<0.05),PBL对自体肿瘤细胞的杀伤活性则明显低于对K562细胞的杀伤活性(P<0.05),SEA激活组比IL-2组杀伤率高(P<0.05);(4)各效应细胞分泌的TNF-a、IFN-γ分别在培养的第2天和第4天达到高峰,高峰后迅速下降,SEA诱导组在前10天明显高于IL-2诱导组(P<0.05)。结论:SEA可高效、迅速诱导卵巢癌TIL的抗瘤活性。     

Influence of paternal lymphocyte immunization on the selected subpopulations of peripheral blood lymphocytes in women with recurrent spontaneous abortions of unknown etiology

The aim of the study was to evaluate the influence of alloimmunization on the composition of PBL subpopulations in women with recurrent spontaneous abortions (RSA) of unknown etiology. MATERIAL AND METHODS: 25 women with history of 3-6 consecutive RSA were selected for this study. Immunization with paternal lymphocytes, isolated from 100 ml of peripheral blood, was performed two times before conception (4-weeks interval). The following parameters were studied: (%) of peripheral T CD3+, T CD4+, T CD8+, T CD4+/CD45-RO+, T CD4+/CD29+, T CD8+/CD45-RO+, BCD19+ and NK CD16+/CD56+ lymphocytes; (%) of T CD3+ cells with markers of early (CD69+) and late activation (HLA-DR+) and presenting with IL-2 receptors (CD25+); the influence of alloimmunization on the expression of CD69+ receptors on cultured T CD3+ lymphocytes activated by PHA (CD3+/CD69+/FCS). RESULTS: The (%) of T CD3+ (61.0% vs. 68.6%), T CD4+ (37.4% vs. 45.3%) cells as well as T CD4+/T CD8+ ratio (1.3 vs. 1.0) significantly increased, whereas the (%) of T CD8+ (41.3% vs. 32.5%) and NK CD16+/CD56+ cells (23.9% vs. 16.2%) decreased after alloimmunization. The procedure didn't influence the (%) of T CD4+/CD45-RO+, T CD4+/CD29+, T CD8+/CD45-RO+ and BCD19+ lymphocytes. We observed significant increase of the (%) of T CD3+/CD69+ (4.4% vs. 7.3%) and T CD3+/CD25+ (5.8% vs. 10.9%) cells after immunotherapy. Moreover we observed the significant increase of the CD69+ expression on T CD3+ cells activated by PHA (CD3+/CD69+/FCS) (42.1 vs. 47.1%). CONCLUSIONS: Our study suggests that alloimmunization of women with RSA of unknown etiology results in increase of T lymphocytes' activity and a shift of the immune balance towards T helper and T supressor lymphocytes.     

HPV16E7肽疫苗对人免疫重建荷人宫颈癌细胞株SiHa细胞SCID鼠免疫功能的影响

目的:观察HPV16E7多肽疫苗对人免疫重建荷人宫颈癌细胞株SiHa细胞严重联合免疫缺陷(SCID)鼠移植瘤的影响。方法:对SCID鼠腹腔注射人外周血淋巴细胞(PBL)后24小时,皮下接种HPV16阳性的人宫颈癌细胞株SiHa细胞,建立人免疫重建荷SiHa细胞的SCID鼠模型,当移植瘤最小体积达100mm3时,三次背部皮下接种疫苗,每次接种间隔二周,观察荷瘤鼠一般生物学特性,检测血浆中人IgG含量、外周血中人CD3+、CD4+、CD8+T细胞、脾重、肿瘤浸润淋巴细胞(TIL)以及脾细胞毒杀伤功能。实验设立空白组,仅为腹腔内注射PBL,疫苗成份仅为不完全佐剂(IFA);随机多肽组,为腹腔内注射PBL,皮下接种SiHa细胞,疫苗成份仅为随机多肽+T辅助多肽+IFA;T辅助多肽组,为腹腔内注射PBL,皮下接种SiHa细胞,疫苗成份仅为T辅助多肽+IFA;多肽治疗组,为腹腔内注射PBL,皮下接种SiHa细胞,疫苗成份为HPV16E711-20(YMLDLQ-PETT),HPV16E786-93(TLGIVCPI)+T辅助多肽+IFA。结果:各组SCID鼠血浆中人IgG水平,8周内随重建时间延长而升高(P<0.05),多肽治疗组达2957.85μg/m l,但各组间无显著性差异;外周血中均可检测到人CD3+、CD4+、CD8+T细胞,但各组间无显著性差异(P>0.05);与空白组比较,其他三组SCID鼠脾重均显著增加(P<0.05)。组织学观察到移植瘤局部TIL浸润及明显的出血和坏死,但经免疫组化未检测到人CD8+T细胞。与空白组比较,多肽治疗组、随机多肽组、辅助多肽组的脾细胞毒杀伤功能均显著降低(P<0.05)。结论:人免疫功能重建的荷SiHa细胞的SCID鼠体内重建的人免疫功能随荷瘤时间的延长受到抑制,多肽疫苗在一定程度上能减轻免疫抑制状态。     

卵巢上皮性癌肿瘤浸润淋巴细胞与腹水中肿瘤相关淋巴细胞生物？…

比较卵巢上皮性癌肿瘤浸润淋巴细胞及腹水中肿瘤相关淋巴细胞的生物学特性。方法收集１２例上皮性卵巢癌组织及腹水 ,并其为实体瘤,血性腹水和非血性腹水３组。将３组标本分别在体外进行ＴＩＬ及ＴＡＬ分离,并在自体肿瘤细以及重组白细胞介素２存在的条件下共同培养。其间     

T lymphocytes and cytokine production in ascitic fluid of ovarian malignancies.

The activation status of T lymphocytes and the presence of various cytokines in ascitic fluid were examined to test peritoneal immunity in women with ovarian malignancies. Peripheral blood and peritoneal fluid were collected from 12 patients with primary ovarian cancer with ascites and 27 normal control subjects during laparoscopic examination. Lymphocyte subpopulations and the expression of activation markers on T lymphocytes were analyzed by dual-color flow cytometry. The concentrations of various cytokines and soluble interleukin (IL)-2 receptor-alpha were measured. CD8 T lymphocytes were the main component of peritoneal lymphocytes. CD69 and HLA-DR, but not CD25, were highly expressed on peritoneal T lymphocytes compared to those in peripheral blood. In ascitic fluid of ovarian malignancies, CD4 T lymphocyte concentrations were further decreased, resulting in a decreased CD4/CD8 ratio. Decreased expression of CD69 and CD25 was also noted on T lymphocytes from ascites compared with T lymphocytes in normal peritoneal fluid. IL-1b, tumor necrosis factor-alpha, IL-6, and soluble IL-2 receptor-alpha concentrations were increased significantly in the ascitic fluid of women with ovarian cancer. The decrease in activation markers on T lymphocytes is suggestive of an immunosuppressive state, despite the presence of abundant stimulatory cytokines. The immunosuppression may be multifactorial, attributed, in part, to the increased concentrations of soluble IL-2 receptor-alpha and other inhibitors.     

子宫颈上皮内病变及子宫颈癌患者外周血及局部免疫功能分析#br#

Objective?To investigate the distribution of T cell subsets in peripheral blood of different cervical lesions and the level of cytokines secreted, and it’s difference in the location and density of immune cell distribution in the epithelium and stroma of cervical lesion tissues. Methods?We used CD series cell detection slide quality control kit to detect the distribution of T cell subsets in peripheral blood of 55 patients with different cervical lesions, analyzed the changes of peripheral blood cytokines, and analyzed the localization and density distribution of T lymphocytes (CD4+T, CD8 +T) in the tissue microenvironment of 64 patients with different cervical lesions by immunohistochemistry. Results?The level of CD4+T and CD8+T in peripheral blood of patients with low grade lesion (LSIL) were lower, with 479.13±229.65 unit and 378.00±231.74 unit respectively. The level of CD4+T and CD8+T in high grade cervical squamous intraepithelial lesion(HSIL)was 816.30±284.65 unit and 668.75±268.92 unit respectively, and the level of CD4+T and CD8+T in carcinoma was 824.80±330.65 unit and 564.00±58.31 unit. Meanwhile the concentration of IL-17 in the serum of LSIL patients was higher than that of HSIL (P<0.05). CD8+T cells in epithelial and stroma tissues of cervical cancer were higher than those in control, LSIL and HSIL tissues, with statistical significance (P<0.05). However, CD4+T cells in the stroma of cancer tissues were higher than those of control, LSIL and HSIL tissues, with statistical significance (P<0.001). Conclusion?The patients with cervical lesion have abnormal immune function in the systemic immunity and local microenvironment of the lesion, both CD8+T cells and CD4+T cells play a key role in eliminating HPV-infected cervical epithelial cells. The development of cervical lesions is related to the cellular inflammatory factors.     

The decidua of early human pregnancy: immunohistochemistry and function of immunocompetent cells.

Like the endometrial stroma, the decidua contains lymphoreticular cells, and these are probably involved in immunological interactions between the conceptus and the mother. Lymphoreticular cells in decidual tissue obtained from 12 patients undergoing therapeutic abortion of an intact pregnancy at 6-10 weeks' gestation were investigated in this study. Immunophenotyping with a broad panel of monoclonal antibodies revealed various subpopulations of lymphoreticular cells. Macrophages (Ki-M6+, Ki-M7+, Ki-M8+, KP1+, MAC 387+ and Ki-M1P+) represented the largest fraction of intradecidual lymphoreticular cells. CD3+ and CD8+ lymphocytes were found in moderate numbers and CD4+ cells in small numbers. The majority of the intradecidual lymphoid cells exhibited an unusual phenotype [CD7+, CD2+, CD56+, triple negative (CD3-, CD4-, CD8)]. The distribution of these unusual lymphocytes mirrored that of the so-called endometrial stromal granulocytes. A few of these stromal granulocytes reacted with the macrophage-associated antibody KP1, but not with Ki-M1P, another macrophage marker. This was confirmed by immuno-electron microscopy. The finding that intradecidual CD3+ lymphocytes express neither the alpha/beta nor the gamma/delta heterodimer of the T cell antigen receptor was unexpected. However, these cells did express the alpha/beta heterodimer after in vitro culture with PHA-P and recombinant exogenous interleukin-2. No stimulated T lymphocytes expressing activation antigens could be detected. B lymphocytes, T and B immune accessory cells and CD15+ granulocytes were found only in small numbers or were absent. Amongst cells expressing NK cell markers, CD57+ and CD16+ cells were found in small to moderate numbers, while CD56+ cells were detected in large numbers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lymphocyte subset distribution and cytokine secretion in third trimester decidua in normal pregnancy and preeclampsia

BACKGROUND: Excessive Th1 activity in peripheral blood plays a probable role in the pathogenesis of preeclampsia. The aim of the study was to investigate whether disturbed local immune reactions are also present in decidua. METHODS: Flow cytometric analysis of CD3, CD19, CD56/CD16, CD4, CD8, CD4/CD29, CD4/CD45RA, CD4/CD45RO, CD8/CD28, CD3/CD69 lymphocyte subsets isolated from third trimester decidua of pregnants with preeclampsia (n=21) and pregnant controls (n=11) subjected to elective caesarean sections. Spontaneous and phytohemaglutynine stimulated "in vitro" secretion of IL-2, IL-4, IL-6, IL-10, IL-12, IFN-gamma and TGF-beta by decidual lymphocytes was studied by ELISA. For the statistical significance of differences between the groups the U Mann-Whitney test was performed (confidence interval P<0.05). RESULTS: Preeclamptic patients were characterized with an increased percentage of the CD3-/CD56+CD16+, CD8+/CD28+ and decreased percentage of CD3+, CD19+, CD4+/CD45RA+ lymphocytes. The profile of secreted cytokines shifts in favor of Th1 activity (extremely high IFN-gamma and low IL-6 and IL-10 secretion). Decidual IL-12 secretion in preeclamptic patients is decreased compared to controls. CONCLUSION: Changes in NK and T lymphocyte subsets followed with Th1 cytokine IFN-gamma over-activity, could affect local immunoregulatory mechanisms in third trimester decidua of preeclamptic patients.     

卵巢上皮性癌肿瘤浸润淋巴细胞分离、培养及生物学特性的研究

目的：分离培养卵巢上皮性癌肿瘤浸润淋巴细胞（ＴＩＬ）,探讨其生物学特性。方法：用酶法或机械法消化肿瘤组织后,在有ＩＬ ２、ＰＨＡ或ＯＫＴ３ 条件下培养。ＴＩＬ表型分析采用间接免疫荧光法,细胞毒活性测定采用ＭＴＴ检测法。结果：分离培养２０ 份卵巢癌ＴＩＬ,１８ 份ＴＩＬ全部活化,另２份污染,最大扩增５２３倍。初分离的卵巢癌ＴＩＬ处于功能抑制状态（抑制期５～１５ｄ）,对Ｋ５６２ 、Ｒａｊｉ和自体瘤细胞的细胞毒活性均很低,经体外培养活化增殖后,细胞毒活性明显增加,以特异性杀伤自体瘤细胞的能力增加最为明显。表型分析表明,ＴＩＬ体外培养过程中,ＣＤ３ 、ＣＤ８、ＣＤ２５（ＩＬ ２Ｒ）和ＨＬＡ ＤＲ的表达率均明显上升。结论：卵巢癌ＴＩＬ体外培养后特异性杀伤自体瘤细胞的能力明显增加,可用于生物学治疗。     

Rapid clearance of herpes simplex virus type 2 by CD8+ T cells requires high level expression of effector T cell functions

CD8(+) T cells are important for resolution of HSV-2 lesions from the female genital epithelium. It is uncertain whether optimal clearance of viruses such as HSV-2 that cause a limited, non-systemic infection solely requires expression of effector functions by infiltrating CD8(+) T lymphocytes, or if the clearance rate is reflective of the expression level of critical effector functions. To address this, CD8(+) T cells from normal OT-I mice or OT-I mice deficient in IFNγ (IFNγ(-/-)) or the IFNγ receptor (IFNγR(-/-)) were activated in vitro in the presence of IFNγ or IL-4 to generate a series of effector populations (Tc1 and Tc2-like respectively) that secreted different levels of IFNγ and expressed different levels of HSV-specific cytolytic function. Compared with Tc1 cells, Tc2-like cells produced the type 2 cytokines IL-4 and IL-5, exhibited decreased IFNγ secretion, diminished proliferation in vitro, and decreased antigen-specific cytolysis in vivo. Clearance of an ovalbumin-expressing HSV-2 strain (HSV-2 tk(-) OVA) by adoptively transferred Tc2-like cells was delayed relative to Tc1 cell recipients. Because donor Tc2-like cells proliferated in vivo and infiltrated the infected genital epithelium similar to Tc1 cells, the diminished virus clearance by Tc2-like effector cells correlated with reduced expression of critical effector functions. Together, these results suggest that high level expression of protective T cell functions by effector T cells is necessary for optimal clearance of HSV-2 from the genital epithelium. These results have important implications for vaccines designed to elicit CD8(+) T cells against viruses such as HSV-2 that infect the genital tract.     

Local immune response in squamous cell carcinoma of the uterine cervix

The role of Langerhans cells as antigen-presenting cells was examined in cervical carcinomas. Frozen samples were obtained from 34 women with stage Ib and II cervical carcinomas. Langerhans cells (CD1), T lymphocytes (CD4 and CD8), B lymphocytes (CD22), and natural killer (CD57, NK) cells were all quantitatively assessed in cervical carcinomas using immunohistochemical methods. These results were related to the MHC class I and II expression on the tumor cells. The majority of Langerhans cells were distributed among cancer cells and they were positively correlated with CD4+, NK and B cells in cervical carcinomas. This is suggestive of the presence of local immune response. The numbers of Langerhans, CD4+, CD8+ and NK cells did not significantly correlate with age at operation, lymph node metastases or depth of cervical wall invasion. The downregulation of MHC class I expression found in 8 (24%) carcinomas was not associated with the decrease in the number of immunologic cells. The upregulation of MHC class II expression found in 26 (76%) carcinomas was significantly associated with the increase in the number of Langerhans cells (p < 0.007). However, the association between the upregulation of MHC-II expression and CD4+ cells did not reach statistical significance (p < 0.07). This is probably due to a small case in this study. MHC-II-restricted immunity may partly contribute to the local immune response in stages Ib and II squamous cell carcinoma of the uterine cervix.     

Presence of an eosinophilic infiltrate in cervical squamous carcinoma results from a type 2 immune response.

OBJECTIVES: The presence of an eosinophilic infiltrate in patients with cervical squamous carcinoma has been shown to correlate with a worse overall survival, suggesting a less effective immune response in these cases. Since type 2 cytokines such as IL-4 and IL-5 are known to attract eosinophilic granulocytes, an immunohistochemical study was performed to gain further insight as to whether a type 1 or type 2 immune response is involved in eliciting an eosinophilic infiltrate. MATERIAL AND METHODS: Frozen tissue sections of 9 normal cervical tissues, 23 premalignant cervical lesions, and 23 cervical squamous carcinomas were stained by immunohistochemistry with monoclonal antibodies directed against IFN-gamma and IL-4 as representatives of a type 1 or a type 2 response, respectively. RESULTS: Normal tissues and premalignant lesions of the cervix did not contain eosinophilic granulocytes and showed very few IL-4- and IFN-gamma-positive cells. In cervical carcinoma the presence of IL-4 on tumor infiltrating cells correlated with the presence of eosinophilic granulocytes in the tumor (P value <0.01) and stroma (P value <0.05). IFN-gamma-positive cells did not show any such correlation. In addition, colocalization was observed of CD3- and IL-4-positive T lymphocytes indicating that IL-4 production is mediated by T lymphocytes. CONCLUSION: The relative increase of IL-4-positive cells in the presence of an eosinophilic infiltrate might thus reflect an imbalance between a type 1 and type 2 response, in favor of the latter. Since a type 1 response stimulates an adequate cellular response which is negatively regulated by type 2 cytokines, these findings might explain the worse clinical outcome seen in cervical cancer patients with an eosinophilic tumor infiltrate. These results may have implications when developing immunotherapeutical strategies for cervical cancer.     

Development, characterization and distribution of adoptively transferred peripheral blood lymphocytes primed by human papillomavirus 18 E7--pulsed autologous dendritic cells in a patient with metastatic adenocarcinoma of the uterine cervix

We describe a 27-year-old woman with systemic chemoresistant and radioresistant metastatic disease secondary to a recurrence of human papillomavirus (HPV) 18 infected cervical adenocarcinoma of the uterine cervix who received adoptive transfer of peripheral blood T cells stimulated with HPV 18 E7-pulsed autologous dendritic cells (DC). Extensive in vitro characterization of the DC-activated T cells derived from peripheral blood mononuclear cells (PBMC) included phenotypic analysis, cytotoxicity and intracellular cytokine production. High cytotoxicity activity was observed by CD8+T cells against autologous tumor cells, but not against NK-sensitive K562 cells, autologous Con-A lymphoblasts, or autologous Epstein-Barr virus-transformed lymphoblastoid cells. Blocking studies demonstrated that lytic activity was significantly inhibited by pretreatment of tumor targets with MAb specific for HLA class I as well as that of effector cells with anti-CD8, anti-LFA-1, but not anti CD3 MAb. Two-color flow cytometric analysis of the cytotoxic T cells revealed that a significant proportion of CD8+ cells was also CD56+. These double positive CTLs were thymically derived, as shown by expression of heterodimeric CD8 molecules (alpha/beta CD8) and were endowed with high cytotoxic activity against tumor cells. Analysis of intracellular cytokine expression showed that the striking majority of E7-pulsed DC activated CD8+ T cells strongly expressed IFN-gamma, TNF-alpha and IL-2 but not IL-4. The patient received two infusions of cytotoxic tumor-specific T cells at 2 week intervals, and in vivo distribution of the T cells was followed by 111 oxine labeling and serial gamma camera imaging. Persistent accumulation of radioactivity in the lungs, which harbored extensive metastatic disease, was detected up to 120 hrs after the infusion. Taken together, these results illustrate the potential of E7-specific and tumor-specific CTL-based immunotherapy for the treatment of patients with invasive cervical cancer.     

Recruitment of CD4 T lymphocytes and macrophages into the cervical epithelium of women after coitus

OBJECTIVE: T lymphocytes and macrophages are considered essential components of the immune response. Many factors are known to influence the presence and distribution these cells in genital mucosa. This study investigated the effect of sexual intercourse on cervical intraepithelial T lymphocytes and macrophages in healthy uninfected women. STUDY DESIGN: Cervical intraepithelial samples were obtained with an endocervical brush from 31 women; the cervical T lymphocytes and macrophages were analyzed by flow cytometry. Eleven women with a history of last sexual intercourse at <3 days were compared against 20 women with last sexual intercourse of >3 days. Furthermore, cellular activation markers (CD69, CD25, HLA-DR) on T lymphocytes and costimulatory molecules (CD80, CD86) on macrophages were studied. RESULTS: Women with last sexual intercourse at <3 days showed predominance of CD4(+) T lymphocytes compared with women with last sexual intercourse of >3 days (P <.02); the numbers of macrophages were higher in the latter (P <.005). No difference was found in the density of T-lymphocyte activation and macrophage costimulatory markers between the two cohorts. CONCLUSION: Within cervical epithelium, the distribution of mononuclear leucocytes may be altered after coitus. The higher proportion of cervical intraepithelial CD4(+) T cells that were observed in the early postcoital period suggests a mechanism by which the relative risk of the acquisition of human immunodeficiency virus infection is increased in women.     

复发性流产外周IL-10~+Tim-3~+ T细胞降调节

目的探讨外周血CD3+T细胞中T细胞免疫球蛋白黏蛋白-3(Tim-3)及程序性死亡因子-1(PD-1)联合细胞因子在复发性流产(RSA)中的诊断价值。方法用流式细胞术检测19例RSA患者及17例正常早孕者外周血CD3~+T细胞表面Tim-3及PD-1含量,以及破细胞膜后细胞内因子干扰素(IFN)-γ和白介素(IL)-10在Tim-3~+PD-1~+、Tim-3~-PD-1~-、Tim-3~-PD-1~+和Tim-3~+PD-1~-4群细胞内的表达情况。结果 RSA患者Tim-3~+PD-1~+T细胞比例(0.57%±0.26%)明显低于早孕组(1.24%±0.77%)(P0.001)。RSA组4群T细胞中IL-10阳性细胞所占比例分别为33.55%±16.27%、0.92%±0.88%、1.61%±1.35%、16.36%±13.98%;早孕组4群T细胞中IL-10阳性细胞占比分别为45.92%±17.89%、0.49%±0.27%、0.92%±0.68%、33.43%±16.98%。RSA组和正常组Tim-3~+PD-1~+T细胞群中IL-10的含量均显著高于其他3群(P0.05);RSA组Tim-3~+PD-1~+和Tim-3~+PD-1~-T细胞中IL-10阳性细胞含量(33.55%±16.27%,16.36%±13.98%)显著低于正常早孕组(45.92%±17.89%,33.43%±16.98%)(P0.05,P0.01)。而IFN-γ在RSA组和正常组4群细胞中的表达无统计学差异。结论 RSA患者IL-10~+Tim-3~+T细胞显著降低,可作为判断RSA的新的参考指标。     

肿瘤浸润淋巴细胞在浆液性卵巢癌的临床病理分析

目的:探讨浆液性卵巢癌中肿瘤浸润性淋巴细胞(TIL)的临床病理和免疫组织化学特征及临床意义。方法:利用免疫组织化学法对68例浆液性卵巢癌中TIL进行组织病理学观察,并分析其与各临床病理因素的关系。结果:68例浆液性卵巢癌中有大量TIL浸润者(≥50 TIL/100肿瘤细胞)43例,占63.24%。有无大量TIL浸润在不同肿瘤细胞分化程度、临床分期、CA125水平中差异有统计学意义(P0.05)。浆液性卵巢癌组织癌巢内CD3~+、CD4~+、CD8~+细胞数显著低于间质内(P0.05);癌巢内CD4~+/CD8~+比值也明显低于间质内(P0.05)。Ⅲ期、低分化组癌巢内及间质内CD4+/CD8~+比值分别低于Ⅰ~Ⅱ期与高-中分化组(P0.05)。癌巢内CD8+/FoxP3~+Treg比值显著低于间质(P0.05);Ⅲ期、低分化组肿瘤癌巢内的CD8+/FoxP3+Treg比值显著降低(P0.05)。Ⅲ期、低分化组患者癌巢中GzmB表达分别低于Ⅰ~Ⅱ期和高-中分化组(P0.05)。结论:有无大量TIL浸润与浆液性卵巢癌肿瘤分化程度、临床分期、CA_(125)水平有关。     

Synthesis of IFN-gamma by CD8(+) T cells is preserved in HIV-infected women with HPV-related cervical squamous intraepithelial lesions

OBJECTIVE: The aim of this study was to investigate whether coinfection with HIV affects the synthesis of Th1 and Th2 cytokines by peripheral blood T cells of women infected with human papillomavirus (HPV). METHODS: Cervical swabs and peripheral blood were obtained from women referred for colposcopy. HPV DNA by Digene's hybrid capture assay, HIV RNA by Roche's Amplicor assay, and cytokine synthesis of T-cell subsets by flow cytometry were assessed. HPV-associated cervical and HIV-associated immune deficiency diseases were staged using the Bethesda System and the Centers for Disease Control criteria, respectively. RESULTS: Patients with HIV and/or HPV infections had lower percentages of IL-2(+) and higher percentages of IL-10(+) T cells than healthy women. Furthermore, women with both virus infections (HIV(+)/HPV(+)) had significantly fewer IL-2(+) CD4(+), IFN-gamma(+) CD4(+), and TNF-alpha(+) CD4(+) T cells than women with HPV infection alone (HPV(+)). Whereas HIV(+) and healthy women had similar numbers of IFN-gamma(+) CD8(+) T cells, HPV(+) women had significantly fewer IFN-gamma(+) CD8(+) T cells than healthy women. CONCLUSION: HIV infection adversely affects the synthesis of Th1 cytokines by CD4(+), but not IFN-gamma synthesis by CD8(+) T cells of women with active HPV infection. The increase in IFNgamma(+) CD8(+) T cells, a phenotype consistent with cytotoxic T lymphocytes, may account for the stable HIV disease of the women studied. However, the increase in IFN-gamma(+) CD8(+) T cells is less likely to be HPV-specific as there was a higher incidence of HPV-related cervical SIL in HIV(+)/HPV(+) women compared with HPV(+) women.     

Induction of ovarian tumor-specific CD8+ cytotoxic T lymphocytes by acid-eluted peptide-pulsed autologous dendritic cells

OBJECTIVE: To evaluate the potential of dendritic cells pulsed with acid-eluted peptides derived from autologous ovarian cancer cells for eliciting a tumor-specific cytotoxic T cell response in women with advanced ovarian cancer. METHODS: CD8+ T lymphocytes derived from peripheral blood mononuclear cells stimulated in vitro with autologous ovarian tumor peptide-pulsed dendritic cells were tested for their ability to induce an HLA class I-restricted cytotoxic T lymphocyte response against autologous tumor cells. To correlate cytotoxic activity by cytotoxic T lymphocytes with T cell phenotype, we used two-color flow cytometric analysis of surface markers and intracellular cytokine expression (interferon-gamma versus interleukin-4). RESULTS: CD8+ cytotoxic T lymphocyte responses against autologous ovarian tumor cells were elicited in three consecutive women who had advanced ovarian cancer. Although cytotoxic T lymphocyte populations from all women expressed strong cytolytic activity against autologous tumor cells, they did not lyse autologous lymphoblasts or Epstein-Barr virus-transformed cell lines, and they showed negligible cytotoxicity against the natural killer-sensitive cell line K-562. Cytotoxicity against the autologous tumor cells was significantly inhibited by anti-HLA class I (W6/32) and anti-HLA-A2 (BB7-2) monoclonal antibodies. CD8+ cytotoxic T lymphocytes expressed variable levels of CD56 and preferentially expressed interferon-gamma rather than interleukin-4. CONCLUSIONS: Peptide-pulsed dendritic cells induced specific CD8+ cytotoxic T lymphocytes that killed autologous tumor cells from women with advanced ovarian cancer. This finding might contribute to the development of active or adoptive immunotherapy for residual or resistant ovarian cancer after standard surgery and cytotoxic treatment.