CD4+CD25+FOXP3+调节性T细胞在肿瘤免疫逃逸中的作用研究进展

CD4^＋CD25^＋FOXP3^＋调节性T细胞是具有特定表型和抑制功能的T淋巴细胞亚群,可通过多种方式抑制抗肿瘤免疫反应从启动到效应的整个阶段,在肿瘤免疫逃逸中起着重要作用.肿瘤患者体内调节性T细胞增多,不仅是一个普遍现象,而且对预后及疗效评价等多方面具有指导意义,是免疫治疗的新的有效靶点.     

Proportion of CD4+CD25+ regulatory T cell is increased in the patients with ovarian carcinoma

Objective: To study the frequency of the CD4⁺CD25⁺ regulatory T cells (Tregs) in the patients with ovarian carcinoma and its possible mechanism. Methods: The percentages of CD4⁺CD25⁺ Tregs in the peripheral blood lymphocytes (PBLs), tumor infiltrating lymphocytes (TILs) and tumor associated lymphocytes (TALs) from 13 patients with ovarian carcinoma and in the PBLs from 14 healthy women were determined by flow cytometry. The expression of CD69 on CD4⁺PBLs from the patients was detected. PBLs from healthy women were cultured in complete RPMI 1640 containing the supernatant from SKOV3 cell line with or without PHA (phytohemagglutinin) stimulation for 72 hours, then the percentage of CD4⁺CD25⁺ T cells was detected. Results: CD4⁺CD25⁺ Tregs in the PBLs from patients with ovarian carcinoma were significantly increased compared with those from the control. The percentage of CD4⁺CD25⁺ Tregs in TILs was higher than that in PBLs and TALs from the patients, but not significantly. The expression of CD69 on CD4⁺PBLs from the patients was negative. The percentages of CD4⁺CD25⁺ T cells in PBLs cultured with SKOV3 supernatant elevated significantly compared with those without supernatant whether PHA was added or not (P = 0.001 and 0.001, respectively). Conclusion: There is an increasing of the proportion of CD4⁺CD25⁺ Tregs in PBLs, TILs and TALs of the patients with ovarian carcinoma, which probably results from up-regulation of soluble factor secreted by ovarian carcinoma cells.     

CD4+CD25+FOXP3+T细胞在人脑星形胶质瘤中的表达和意义

目的：观察调节性T细胞在星形胶质瘤患者体内的表达情况, 观察其与星形胶质瘤发展的关系。方法： 收集河北医科大学第四医院神经外科在2008年9月至2010年6月期间收治的星形胶质瘤患者, 共92例, 其中男性54例, 女性38例, 平均年龄51.4岁, 均为初次发病。收集8例行颞叶切除术的癫痫患者为对照组。应用流式细胞术检测观察CD4+CD25+FOXP3+调节性T细胞 （Treg） 在胶质瘤组织中及患者外周血中的浸润情况, 应用免疫酶联反应方法 （ELISA） 测定转化生长因子β1 （TGFβ1） 在外周血中的表达, 应用SPSS13.0软件进行统计学分析处理。结果： 星形胶质瘤患者外周血 （9.34±2.13） %和肿瘤组织 （24.17±2.54） %中的Treg细胞明显增多, 均显著高于对照组 （0.67%±0.12%, 24.17%±2.54%, P<0.05）, 并且Treg细胞的含量随着肿瘤级别的升高而增加, WHO Ⅳ级患者外周血和肿瘤组织中的Treg细胞含量 （8.34%±2.13%, 35.45%±2.47%） 明显高于WHO Ⅲ和WHO Ⅱ级 （P<0.05）。ELISA结果显示： 星形胶质瘤患者外周血中TGFβ1的含量明显增高, 并与外周血中的Treg细胞的数量明显呈正相关 （r=0.564, P=0.001）。结论： Treg细胞在星形胶质瘤患者的外周血和肿瘤组织的浸润明显增多, 并与肿瘤的恶性程度有关, 其数量的增长与TGFβ1的表达相关。Treg细胞的增多能够抑制机体抗肿瘤免疫, 促进了星形胶质瘤的发展, 其具有成为胶质瘤免疫治疗靶点的潜在价值。     

Disease stage variation in CD4+ and CD8+ T-cell reactivity to the receptor tyrosine kinase EphA2 in patients with renal cell carcinoma

We have evaluated CD8+ and CD4+ T-cell responses against a new tumor-associated antigen, the receptor tyrosine kinase EphA2, which is broadly expressed in diverse cancer histologies and is frequently overexpressed in advanced stage/metastatic disease. We report herein that EphA2 is overexpressed in renal cell carcinoma (RCC) cell lines and clinical specimens of RCC, and find that the highest levels of EphA2 are consistently found in the most advanced stages of the disease. We identified and synthesized five putative HLA class I-binding and three class II-binding peptides derived from EphA2 that might serve as targets for immune reactivity. Each peptide induced specific, tumor-reactive CD8+ or CD4+T-cell responses as measured using IFN-gamma enzyme-linked immunospot assays. The EphA2 peptides elicited relatively weak responses from CD8+ T cells derived from normal healthy volunteers or from RCC patients with active disease. In marked contrast, immune reactivity to EphA2-derived epitopes was greatly enhanced in CD8+ T cells that had been isolated from patients who were rendered disease-free, after surgery. Furthermore, enzyme-linked immunospot analyses demonstrated prominent EphA2-restricted T-helper 1-type CD4+ T cell activity in patients with early stage disease, whereas T-helper 2-type and T regulatory-type responses predominated in patients with more advanced forms of RCC. These data suggest that the immune system of cancer patients actively monitors EphA2-derived epitopes, and that the magnitude and character of T-cell responses to EphA2 epitopes may convey much-needed predictive information about disease stage and outcome.     

The effects of trastuzumab on the CD4+CD25+FoxP3+ and CD4+IL17A+ T-cell axis in patients with breast cancer

In addition to the direct targeting effects on HER2-positive cells, trastuzumab may have a therapeutic role modulating the activity of the cellular immune system in patients with breast cancer. To investigate this further, the balance of T-regulatory (T_reg), Th17, natural killer (NK) and NK T (NKT) cells before, during and after trastuzumab therapy was investigated. Sequential frequencies of circulating T_reg cells, Th17 cells, NK and NKT cells were measured in peripheral blood of breast cancer patients and normal controls throughout therapy. Individuals with breast cancer had significantly higher T_reg frequencies of peripheral blood compared with healthy controls (9.2 or 8.6 vs 6%; P<0.05), and no significant differences in T_reg frequencies were observed between HER2-positive and HER2-negative individuals. The number of Th17 cells was lowest in HER2-positive patients compared with both healthy controls and HER2-negative patients (0.31 vs 0.75% or 0.84%; P=0.01). There appeared to be an inverse relationship between T_reg and Th17 frequencies in metastatic breast cancer (MBC) with T_reg levels significantly reduced during treatment with trastuzumab (P=0.04), whereas Th17 frequencies were concomitantly increased (P=0.04). This study supports earlier data that T_reg cells are present at higher frequencies in breast cancer patients compared with healthy individuals. For the first time, we show that HER2-positive individuals with breast carcinomas have reduced numbers of circulating Th17 cells, which appear, in turn to have an inverse relationship with T_reg frequency in MBC. The change in balance of the T_reg : Th17 ratio appears to characterise the cancer state, and furthermore, is disrupted by trastuzumab therapy.     

胃癌患者外周血中CD4~+CD25~(high)FOXP3~+调节性T细胞的检测及临床意义

目的探讨胃癌患者外周血CD4+CD25highFOXP3+调节性T细胞(Treg)的检测及临床意义。方法选取60例胃癌患者(胃癌组)和60例健康体检者(健康对照组),采用流式细胞仪检测外周血中CD4+CD25highFOXP3+Treg细胞、CD4+CD25highT细胞及CD4+T细胞水平,酶联免疫吸附法检测外周血白介素10(IL-10)和转化生长因子β1(TGF-β1)浓度。结果与健康对照组相比,胃癌组患者外周血中CD4+CD25highFOXP3+Treg细胞和CD4+CD25highT细胞比例增高;IL-10和TGF-β1浓度增高,差异均有统计学意义(均P<0.05)。结论 CD4+CD25highFOXP3+Treg细胞可能与胃癌发生发展密切相关,为胃癌免疫治疗提供新思路。     

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。     

Skewed immunological balance between Th17 (CD4+IL17A+) and Treg (CD4+CD25+FOXP3+) cells in human oral squamous cell carcinoma

Background

Several studies have documented modulation of Th17 and T regulatory (Treg) cells in various human malignancies which may vary with the type and extent of the disease. However, such data in patients with oral cancer is scarce and hence the current study was designed to elaborate the immunological balance between these two T cell subsets in oral cancer.

Methods and results

We analyzed various T cell subsets in the peripheral blood of 45 oral squamous cell carcinoma (OSCC) patients and 40 healthy volunteers. We found that, compared with the healthy controls, patients had a significantly (p?<?0.0001) higher proportion of both Th17 (CD4⁺IL17A⁺) and Treg (CD4⁺CD25⁺FOXP3⁺) cells, which further showed a reciprocal balance in relation to clinico-pathological parameters in patients. We also detected a circulating CD8⁺ subset of these cells in both patients and healthy controls, although the difference between the two groups was statistically insignificant. Higher frequencies of Th17 cells were found in patients with early stages and without lymph node involvement, while an increased prevalence of Tregs was associated with higher clinical stages and lymph node involvement. Moreover, Th17 cells were quantitatively and positively correlated to CD4⁺T and CD8⁺T cells and inversely correlated with Tregs. Contrarily, Tregs showed a negative association with CD4⁺T and CD8⁺T cells.

Conclusions

Our results suggest an increase in Th17/Tregs ratio in early stages and a decrease in this ratio in higher stages of oral cancer. Such counter regulation of Th17 and Tregs may be a significant prognostic factor in oral cancer patients.     

Identification of HNP3 as a tumour marker in CD4+ and CD4- lymphocytes of patients with cutaneous T-cell lymphoma

Cutaneous T-cell lymphomas (CTCL) are characterized by malignant proliferation of skin homing T-cells. Although prognosis is generally good, reliable markers are needed to identify patients at risk for a more aggressive course. ProteinChip (SELDI) technology was used as a tool for the discovery of protein patterns in lymphocytes from patients with CTCL (n=25) and unaffected controls (n=25). Lymphocytes were separated in CD4+ and CD4- fractions by magnetic cell sorting (MACS). Each whole protein extract was analysed by ProteinChip technology. The resulting protein profiles were submitted for bioinformatic analysis including a clustering algorithm, a rule extraction, a rating and a rule-base construction step. For the generated combined rule base for the CD4- cell fraction, both the sensitivity and specificity for the prediction of CTCL reached 96%, while for the CD4+ fraction they were 92% and 84%, respectively, for sensitivity and specificity. The most significant peak at 3489Da could be identified as HNP3, an alpha-defensin, by immunocapturing. These results open up both the possibility for the use of this protein signature, especially HNP3, to more effectively monitor and screen CTCL, and the avenue to identify the other relevant peaks for a better understanding of the development of this tumour.     

CD4+CD25+ regulatory T-cell-inactivation in combination with adenovirus vaccines enhances T-cell responses and protects mice from tumor challenge

Cancer vaccines are a promising approach to treating tumors or preventing tumor relapse through induction of an immune response against tumor-associated antigens (TAA). One major obstacle to successful therapy is the immunological tolerance against self-antigens which limits an effective antitumor immune response. As a transient reduction of immunological tolerance may enable more effective vaccination against self-tumor antigens, we explored this hypothesis in a CEA tolerant animal model with an adenovirus expressing CEA vaccine in conjunction with inactivation of CD4(+)CD25(+) regulatory T cells. This vaccination modality resulted in increased CEA-specific CD8(+), CD4(+) T cells and antibody response. The appearance of a CD4(+) T-cell response correlated with a stronger memory response. The combined CD25(+) inactivation and genetic vaccination resulted in significant tumor protection in a metastatic tumor model. Non-invasive tumor visualization showed that not only primary tumors were reduced, but also hepatic metastases. Our results support the viability of this cancer vaccine strategy as an adjuvant treatment to prevent tumor relapse in cancer patients.     

An increase in CD4+CD25+FOXP3+ regulatory T cells in tumor-infiltrating lymphocytes of human glioblastoma multiforme

The subpopulation of CD4+CD25+ immunoregulatory T (Tr) cells constitutes 5%-10% of CD4+ cells in humans. These cells play a crucial role in the control of tumor immune response. In this study, we evaluated the distribution of Tr cells in tumor-infiltrating lymphocytes of human glioblastoma multiforme and examined the difference between the brain and autologous blood with respect to Tr cells. Glioma samples from 10 patients were classified as WHO grade IV astrocytoma. Control samples were obtained from patients undergoing resection of a seizure focus. The samples were analyzed by flow cytometry to determine the frequency of Tr cells and by real-time PCR for forkhead box P3 (FOXP3) expression. We then examined the expression of CD62L, CD45RO, and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and assessed the functionality of Tr cells in vitro. There was a significant difference in the number of FOXP3-expressing CD4+CD25+ T cells within glioma-infiltrating lymphocytes as compared to controls (P < 0.01). This difference was further observed in studies of autologous patient blood and control blood. The expression level of FOXP3 mRNA was high in Tr cells and weak in CD4+CD25-T cells. Moreover, the expression of CD62L and CTLA-4 was elevated in glioma Tr cells as compared to that in the controls. These cells were also CD45RO positive. Functional assays confirmed the suppressive activity of Tr cells in patients with glioma. The expression of CD4+CD25+FOXP3+ T cells was significantly higher in patients with glioblastoma multiforme than in controls. This increase in the frequency of Tr cells that display suppressive activity might play a role in modulation of the immune response against glioma. In light of these findings, Tr cells may represent a potential target for immunotherapy of malignant brain tumors.     

Identification of an enriched CD4+ CD8α++ CD8β+ T-cell subset among tumor-infiltrating lymphocytes in human renal cell carcinoma

Three-color immunofluorescence flow-cytometric analysis of freshly isolated tumor-infiltrating lymphocytes (TIL) from patients with primary renal cell carcinoma (RCC) revealed a unique, not previously described TIL subset with a CD3₊ CD4₊ CD8α₊₊ CD8β₊ phenotype. This subset represented at least 5% of CD3₊ TIL in 15 of 21 patients with clear cell RCC, whereas it was not or only marginally represented in patients with papillary RCC or sarcomatoid RCC. In one-third of the patients with clear cell RCC, more than 20% of CD3₊ TIL and in one patient more than half of the CD3₊ TIL displayed this phenotype. The occurrence of this subset was not associated with pathological stage, tumor diameter, nuclear grade, cytogenetic abnormalities or vascular invasion in this patient cohort. When present, the CD3₊ CD8α₊₊ CD8β₊ subset was detected in similar proportions in tumor tissue and tumor capsula, and it was also detected in adjacent non-tumoral renal tissue, albeit in much lower proportions. Despite strong cell surface expression of various activation markers (CD69, CD54 and HLA-DR), CD3₊ CD4⁺ CD8α₊₊ anti-CD3-redirected cytotoxicity assay. In contrast with CD3₊ CD4₊ CD8₋ cells from the same tumor sample, they were markedly deficient in IL-2Rα up-regulation following anti-CD3 triggering. The possibility that these cells represent either anergic cells or a highly specialized effector population with a discrete, as yet undescribed function is discussed. Int. J. Cancer 71:178–182, 1997. © 1997 Wiley-Liss, Inc.     

CD4+ T-cell help in the tumor milieu is required for recruitment and cytolytic function of CD8+ T lymphocytes

CD4 help for CD8(+) T lymphocytes prevents tolerance and promotes the survival of effector and memory CD8(+) T cells. Here, we describe additional helper functions that require CD4(+) T cells within the tumor environment. CD8(+) T-cell recruitment, proliferation, and effector function within the tumor were greatly enhanced by tumor-specific CD4(+) T cells. Recruitment of CD8(+) T cells was accelerated by IFN-γ-dependent production of chemokines. Production of interleukin-2 by tumor resident CD4(+) T cells enhanced CD8(+) T-cell proliferation and upregulated expression of granzyme B. These results highlight a novel role for tumor-specific CD4(+) T cells in promoting CD8(+) T-cell recruitment and cytolytic function, two previously unappreciated aspects of tumor-specific CD4 help.     

肝癌患者CD4+CD25+调节性T细胞的检测及其临床意义

背景与目的CD4 CD25 调节性T细胞(CD4 CD25 regulatoryTcells)在自身免疫耐受、移植和抗肿瘤应答中起着重要的作用,它们在肿瘤患者中的作用机制尚未阐明。本研究分析、探讨肝癌患者肿瘤组织和外周血CD4 CD25 调节性T细胞频率的临床意义。方法应用流式细胞仪分析肝癌患者的肿瘤组织、外周血和非肝癌对照组中单个核细胞(PBMCs)的CD4 CD25 调节性T细胞频率。结果肝癌患者外周血CD4 CD25 T细胞占PBMCs(6.9±2.8)%,肝癌组织CD4 CD25 T细胞占(6.7±1.6)%,均较对照组显著增高。并且CD4 CD25 T细胞的阳性率与淋巴结转移率和TNM分期存在相关性。淋巴结转移阳性和TNM分期越晚,CD4 CD25 T细胞的阳性率越高。结论CD4 CD25 调节性T细胞在肝癌患者中比例升高,可能是肝癌患者细胞免疫功能削弱的机制之一。     

卵巢癌腹水CD4+ CD25+调节性T细胞免疫抑制功能及机制的研究

  目的  研究卵巢癌患者腹水内CD4+ CD25+调节性T细胞（Treg）免疫抑制功能与患者临床病理特点的关系及其与患者初治及复发状态是否相关,并进一步探讨卵巢癌腹水内CD4+ CD25+调节性T细胞（Treg）发挥免疫调节性作用的具体机制。  方法  应用免疫磁珠分选法分选28例卵巢癌患者腹水内CD4+ CD25+调节性T细胞及CD4+ CD25- T细胞,采用羧基荧光素二醋酸盐琥珀酰亚胺酯（carboxyfluorescein succinimidylester,CFSE）标记CD4+ CD25- T细胞,CD4+ CD25+ Treg与自体CFSE标记CD4+ CD25- T细胞以1:1比例共培养,经流式检测CFSE表达及Modfit软件分析CD4+ CD25- T增殖指数（PI）,计算各标本内Treg对自体CD4+ CD25- T细胞增殖抑制率。应用中合性抗IL-10抗体及中合性抗TGF-β1抗体探究腹水内CD4+ CD25+ Treg介导免疫逃逸作用是否通过抑制性细胞因子IL-10或TGF-β1发挥作用。  结果  Ⅲ~Ⅳ期卵巢癌腹水内CD4+ CD25+ Treg对自体CD4+ CD25- T细胞增殖抑制率为（75.72±17.04）%,较Ⅰ~Ⅱ期Treg抑制率（59.61±16.97）%显著增强（P < 0.05）。复发卵巢癌患者腹水内Treg对自体CD4+ CD25- T细胞增殖抑制率为（85.89±7.07）%,较初治卵巢癌患者腹水Treg抑制率（52.82±8.18）%显著增强（P=0.000 1）。共培养体系内加入中合性抗IL-10抗体或/及中和性抗TGF-β1抗体,Treg对自体CD4+ CD25- T细胞增殖抑制率较对照组均显著降低（P < 0.05）。  结论  卵巢癌腹水内CD4+ CD25+ Treg介导免疫逃逸能力与肿瘤分期及复发、初治状态相关,且发挥免疫逃逸作用可能是与分泌抑制性细胞因子IL-10及TGF-β1有关。      

Proliferation of CD4+CD25high+Foxp3+ regulatory T lymphocytes in ex vivo expanded ascitic fluid from primary and recurrent ovarian carcinoma

Objective

Regulatory T lymphocytes evoke the immune tolerance by suppressing and inactivating cytotoxic T lymphocytes. The objective of this study was to compare the proportion of regulatory T lymphocytes, precisely defined as CD4⁺CD25^high+Foxp3⁺ T lymphocytes, in primary and recurrent ovarian carcinoma before and after ex vivo expansion of ascites with interleukin-2 (IL-2).

Methods

Ascitic fluid samples were obtained from 26 patients with ovarian carcinoma. Lymphocytes were isolated from ascites and cell markers were analyzed by flow cytometry using anti-CD3/CD4/CD8/CD16/CD56/CD25 and anti-Foxp3 antibodies. Lymphocytes were incubated for 2 to 3 weeks and expanded ex vivo by IL-2 stimulation and their phenotypes were analyzed by flow cytometry.

Results

Following ex vivo expansion, ascitic fluid lymphocytes increased by a greater extent in the recurrent group than in the primary group. The proportion of ex vivo-expanded lymphocytes changed as follows; CD4⁺ T lymphocytes increased, CD8⁺ T lymphocytes decreased, and the proportion of CD3^-CD16⁺56⁺ NK cells was unchanged. The proportion of CD4⁺CD25^high+Foxp3⁺ regulatory T lymphocytes in CD4⁺ T lymphocytes increased after ex vivo expansion in both groups, but to a greater degree in the recurrent group.

Conclusion

This study showed that regulatory T lymphocytes, neither cytotoxic T lymphocytes nor NK cells, were extensively increased after ex vivo expansion, especially in recurrent ovarian carcinoma. These results may provide information that helps to guide the future development of adoptive immunotherapy against ovarian carcinoma.