肺癌患者胸腔积液及外周血CD4+CD+25 T细胞、Foxp3基因表达及意义

目的探讨CD+4CD+25T细胞及Foxp3基因在肺癌发生、发展中的作用及机制.方法流式细胞仪检测32例肺癌合并胸腔积液患者外周血、胸腔积液及30例无胸腔积液肺癌患者外周血、胸腔冲洗液中CD+4 CD+25T细胞水平,RT-PCR法检测特异性转录因子Foxp3基因在胸腔积液及外周血中的表达.结果肺癌合并胸腔积液患者胸腔积液中CD+4CD+25T细胞水平显著高于无胸腔积液患者胸腔冲洗液及外周血;Foxp3基因在肺癌患者外周血和胸腔积液中呈高表达.结论肺癌患者外周血和胸腔积液中增高的CD+4CD+25T细胞主要是CD+4CD+25调节性T细胞.CD+4cD+25调节性T细胞可促进肺癌的进一步恶化,其机制可能是抑制肿瘤免疫.     

结核性胸膜炎患者胸液中CD4~+ CD25~+ FoxP3~+调节T细胞抑制抗结核免疫

目的研究结核性胸膜炎患者胸液中CD4+CD25+FoxP3+调节T细胞是否增多,这些调节T细胞是否抑制结核的特异细胞免疫反应。方法使用细胞分离、流式细胞分析及体外细胞培养作细胞增殖及增殖抑制等实验方法,对15例结核性胸膜炎患者及17例健康正常人群胸液及外周血白细胞中CD4+CD25+FoxP3+调节T细胞的量及特征作研究。结果结核性胸膜炎患者胸液中CD4+CD25+FoxP3+调节T细胞明显高于患者及健康人群外周血。在体外,结核性胸膜炎患者胸液中单核细胞对BCG刺激产生γ-干扰素(IFN-γ)的能力明显强于患者及健康人群外周血中单核细胞;把这些调节T细胞从胸液单核细胞中清除,增强了结核患者胸液单核细胞对BCG刺激产生IFN-γ;从结核患者胸液分离的这些调节T细胞能抑制结核患者Th1细胞产生IFN-γ。结论结核性胸膜炎患者胸液CD4+CD25+FoxP3+调节T细胞增多,抑制结核性胸膜炎患者Th1细胞免疫反应,从而参与了结核性胸膜炎的发病。     

Pleural effusion of a second neoplasm in a patient with B-CLL: two immunological compartments

We report the case of a patient with a chronic lymphocytic leukemia (CLL) who later developed a metastasized large-cell neuroendocrine carcinoma of the lung that was complicated by a malignant pleural effusion. In contrast to the peripheral blood where the malignant B-CLL cells represented >99% of all lymphocytes, lymphocytes infiltrating the malignant effusion were mainly T cells. Nearly all of these T cells were CD4(+). This stood in sharp contrast to the peripheral blood where the CD4(+)/CD8(+) ratio remained balanced. A detailed analysis of the CD4(+) T cells within the malignant effusion revealed that these cells uniformly expressed a CCR7(+) CD62L(+) "non-effector" phenotype. When the monoclonal B cells within the malignant effusion were analyzed, we found that these cells, in contrast to the B-CLL cells in the peripheral blood, were negative for CD23 and expressed much higher levels of the adhesion molecules L-selectin (CD62L) and CD11a. A deficient expression of these adhesion molecules might have led to a "trapping" of the majority of B-CLL cells in the peripheral blood. This phenomenon might have contributed to the development of two highly different immunological compartments in this patient with CLL and pleural effusion of a solid tumor.     

Apoptosis-induced decrease of intrathyroidal CD4(+)CD25(+) regulatory T cells in autoimmune thyroid diseases.

BACKGROUND: We previously showed that the proportion of CD4(+) T cells was lower and both the proportion and intensity of Fas expression on intrathyroidal CD4(+) T cells were higher in the thyroid than in the peripheral blood of patients with autoimmune thyroid disease (AITD). OBJECTIVE: To clarify whether the intrathyroidal CD4(+)CD25(+) regulatory T (Treg) cells are decreased by Fas-mediated apoptosis in patients with AITD. DESIGN: We examined intrathyroidal and peripheral lymphocytes in 20 patients with AITD (15 patients with Gravesa disease and five patients with Hashimotoas disease) and peripheral lymphocytes in 10 healthy volunteers by three-color flow cytometry. MAIN OUTCOME: The proportion of CD4(+)CD25(+) cells was lower in the thyroid of patients with AITD than in the peripheral blood of the same patients or the peripheral blood of the healthy subjects. The proportions of CD4(+)CD25(+)CD69() cells and Forkhead box P3 (Foxp3)(+)CD4(+)CD25(+) cells, which constitute more specific Treg subsets than CD4(+)CD25(+) cells, were also lower in the thyroid than in the peripheral blood of patients with AITD. The proportion of apoptotic cells was higher among intrathyroidal CD4(+) cells than among peripheral CD4(+) cells and higher among intrathyroidal CD4(+)CD25(+) cells than among intrathyroidal CD4(+)CD25() cells. CONCLUSION: These results indicate that intrathyroidal Treg cells are decreased in response to apoptosis in patients with AITD. This decrease in Treg cells may contribute to the incomplete regulation of autoreactive T cells in AITD.     

CD70+ non-Hodgkin lymphoma B cells induce Foxp3 expression and regulatory function in intratumoral CD4+CD25 T cells

Foxp3 expression was initially thought to be restricted to the CD4(+)CD25(+) regulatory T-cell population. However, recent studies suggest that forkhead box P3 (Foxp3) is expressed in CD4(+)CD25(-) T cells in aged mice. In the present study in B-cell non-Hodgkin lymphoma (NHL), we found that a subset of intratumoral but not peripheral blood CD4(+)CD25(-) T cells, comprising about 15% of intratumoral CD4(+) T cells, express Foxp3 and are capable of suppressing the proliferation of autologous infiltrating CD8(+) T cells. In vitro activation with OKT3/anti-CD28 antibody (Ab) or dendritic cells (DCs) induced Foxp3 expression in a subset of these CD4(+)CD25(-)Foxp3(-) T cells. We found that the presence of lymphoma B cells during activation augmented activation-induced Foxp3 expression in CD4(+)CD25(-) T cells. We also found that CD70(+) lymphoma B cells significantly contributed to the activation-induced Foxp3 expression in intratumoral CD4(+)CD25(-) T cells. Furthermore, the blockade of CD27-CD70 interaction by anti-CD70 Ab abrogated lymphoma B-cell-mediated induction of Foxp3 expression in intratumoral CD4(+)CD25(-) T cells. Taken together, these studies reveal a novel role for NHL B cells in the development of intratumoral regulatory T cells.     

Efficient generation of human alloantigen-specific CD4+ regulatory T cells from naive precursors by CD40-activated B cells

CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg) play an important role in the induction and maintenance of immune tolerance. Although adoptive transfer of bulk populations of Treg can prevent or treat T cell-mediated inflammatory diseases and transplant allograft rejection in animal models, optimal Treg immunotherapy in humans would ideally use antigen-specific rather than polyclonal Treg for greater specificity of regulation and avoidance of general suppression. However, no robust approaches have been reported for the generation of human antigen-specific Treg at a practical scale for clinical use. Here, we report a simple and cost-effective novel method to rapidly induce and expand large numbers of functional human alloantigen-specific Treg from antigenically naive precursors in vitro using allogeneic nontransformed B cells as stimulators. By this approach naive CD4(+)CD25(-) T cells could be expanded 8-fold into alloantigen-specific Treg after 3 weeks of culture without any exogenous cytokines. The induced alloantigen-specific Treg were CD45RO(+)CCR7(-) memory cells, and had a CD4(high), CD25(+), Foxp3(+), and CD62L (L-selectin)(+) phenotype. Although these CD4(high)CD25(+)Foxp3(+) alloantigen-specific Treg had no cytotoxic capacity, their suppressive function was cell-cell contact dependent and partially relied on cytotoxic T lymphocyte antigen-4 expression. This approach may accelerate the clinical application of Treg-based immunotherapy in transplantation and autoimmune diseases.     

Foxp3+CD4+CD25+ T cells control virus-specific memory T cells in chimpanzees that recovered from hepatitis C

Hepatitis C virus (HCV) poses a global health problem because it readily establishes persistent infection and a vaccine is not available. CD4(+)CD25(+) T cells have been implicated in HCV persistence because their frequency is increased in the blood of HCV-infected patients and their in vitro depletion results in increased IFN-gamma production by HCV-specific T cells. Studying a well-characterized cohort of 16 chimpanzees, the sole animal model for HCV infection, we here demonstrate that the frequency of Foxp3(+)CD4(+)CD25(+) regulatory T cells (T(Regs)) and the extent of suppression was as high in spontaneously HCV-recovered chimpanzees as in persistently HCV-infected chimpanzees. Foxp3(+)CD4(+)CD25(+) T(Regs) suppressed IFN-gamma production, expansion, and activation-induced cell death of HCV-specific T cells after recovery from HCV infection and in persistent HCV infection. Thus, T(Reg) cells control HCV-specific T cells not only in persistent infection but also after recovery, where they may regulate memory T-cell responses by controlling their activation and preventing apoptosis. However, Foxp3(+)CD4(+)CD25(+) T(Reg) cells of both HCV-recovered and HCV-infected chimpanzees differed from Foxp3(+)CD4(+)CD25(+)T(Reg) cells of HCV-naive chimpanzees in increased IL-2 responsiveness and lower T-cell receptor excision circle content, implying a history of in vivo proliferation. This result suggests that HCV infection alters the population of Foxp3(+)CD4(+)CD25(+) T(Reg) cells.     

Proliferation and foxp3 expression in virus-specific memory CD8+ T lymphocytes

Foxp3 plays a critical role in development of CD4+ regulatory T lymphocytes (Tregs). It was originally proposed as a specific marker for Tregs, but recent studies have shown that Foxp3 can be expressed in proliferating CD8+ and CD4+ T lymphocytes. We further investigated the association between Foxp3 expression and proliferation of peripheral blood CD4+ and CD8+ T lymphocytes and focused on virus-specific memory CD8+ T lymphocytes. We found that resting peripheral blood bulk and cytomegalovirus- or HIV-1-specific CD8+ T lymphocytes do not normally express Foxp3. However, stimulation in vitro triggered these cells to express Foxp3 as well as CD25, and the addition of interleukin-2 possibly enhanced the expression of Foxp3. These data demonstrate that proliferation itself is sufficient to induce the Treg-like phenotype. Given that others have demonstrated Treg functional activity in such "induced Tregs," these results suggest that virus-specific CD8+ T lymphocytes have the capacity to acquire regulatory functions. Although the implications of Foxp3 expression in virus-specific CD8+ T lymphocytes in the immunologic control of persistent HIV-1 viremia remain to be determined, our results are consistent with Foxp3 expression playing an essential role in regulation of cell proliferation and functional outcomes for HIV-1-specific CD8+ T lymphocytes.     

CD4＋CD25＋调节T细胞在平阳霉素胸膜固定术中的意义

目的探讨外周血及胸水CD4＋CD25＋调节T细胞（Treg）在平阳霉素（PYM）胸膜固定术中的变化及临床意义。方法23例肺癌并发恶性胸腔积液（MPE）患者给予PYM胸膜固定术,对固定术前及术后48h外周血及胸水采用流式细胞术观察Treg变化。结果23例患者治疗4周后,有效14例,无效9例,有效率60．87％。有效组男11例,女3例,无效组男6例,女3例,两组给药前年龄、KPS评分、Treg在血及胸水中比较均无差异。术后患者血及胸水Treg较术前明显减低（血Treg：P=0．019;胸水Treg：P=0．039）。有效组给药后血Treg降低显著（P=0．010）,胸水Treg降低不显著（P=0．092）;无效组血和胸水Treg降低均不明显。结论PYM胸膜固定术可有效控制MPE,其疗效与PYM显著减少血Treg,促进炎症反应有关。     

Significance of the frequency of CD4+CD25+CD127- T-cells in patients with pulmonary tuberculosis and diabetes mellitus

Background and objective: Pulmonary tuberculosis and diabetes mellitus (DM) are closely associated. The objective of this study was to determine whether the expression of CD4+CD25+CD127? T‐cells (regulatory T‐cells (Treg)) is associated with diabetic pulmonary tuberculosis. Methods: Flow cytometry was used to determine the frequencies of CD4+CD25+ and CD4+CD25+CD127? T‐cells in peripheral blood, bronchoalveolar lavage fluid (BALF) and pleural effusions from 120 patients (30 with pulmonary tuberculosis and DM (TBDM), 30 with pulmonary tuberculosis without DM (TB), 30 with tuberculous pleurisy without DM (TBP) and 30 healthy volunteers). The concentrations of interferon (IFN)‐γ and interleukin (IL)‐10 in BALF and pleural effusions were determined by enzyme‐linked immunosorbent assay. Results: Treg frequencies in peripheral blood were significantly higher in patients with TBDM, TB and TBP than in the control group, with the frequency in TBDM being the highest (P < 0.01 for all). In TBP patients, Treg frequencies were significantly lower in pleural effusions than in peripheral blood. In TB patients, Treg frequencies in BALF and peripheral blood were not significantly different. However, in TBDM patients, Treg frequencies were significantly higher in BALF than in peripheral blood. IL‐10 expression was significantly higher, and IFN‐γ expression was significantly lower in BALF of TBDM patients compared with BALF and pleural effusions of TB patients. Conclusions: In patients with pulmonary tuberculosis and DM, the imbalance between Treg and effector T‐cells at pathological sites may be associated with weakened immunity and clinical manifestations of TB.     

Increased population of CD4(+)CD25(high), regulatory T cells with their higher apoptotic and proliferating status in peripheral blood of acute myeloid leukemia patients

PURPOSE: Regulatory T cells (T-reg) that control harmful autoimmune T cells in the periphery may also suppress the immune response against cancer. In this study we investigated the possible involvement of CD4(+)CD25(high) T-reg in the immune impairment of patients with acute myeloid leukemia (AML). EXPERIMENTAL DESIGN: The frequencies and phenotypes of CD4(+)CD25(high) T cells in the peripheral blood of AML patients were determined by flow cytometry. To assess the functional activity of CD4(+)CD25(high) T cells, CD4(+)CD25(high), and CD4(+)CD25(-) T cells were sorted from peripheral blood mononuclear cells with FACS Vantage. The immunoregulatory properties of CD4(+)CD25(high) and CD4(+)CD25(-) T cells were characterized by proliferation assays and cytokine production assays. In addition, the frequency of apoptotic and proliferating cells in CD4(+)CD25(high) T cells were respectively evaluated by 7AAD and ki67 binding cells using flow cytometry. RESULTS: Compared with healthy controls, AML patients had a higher proportion of CD4(+)CD25(high) T cells in peripheral blood. These cells were CD45-RA(-), CD69(-), CD45-RO(+), CD95(+), and intercellular CTLA-4(+), and secreted low levels of TNF-alpha and IL-10, but no IL-2, IL-4, IL-5, and IFN-gamma. They inhibited the proliferation and cytokine production (IL-2, IFN-gamma) of CD4(+)CD25(-) T cells, but improved IL-10 production under the co-culture of both subsets with stimulation, thus behaving as T-reg. Notably, CD4(+)CD25(high) T cells in AML patients presented significantly higher apoptosis and proliferation than that of healthy individuals. CONCLUSIONS: The frequency of CD4(+)CD25(high) T-reg in peripheral blood in AML patients is significantly higher when compared with healthy individuals, likely due to the increasing proliferation of CD4(+)CD25(high) T cells.     

Graves病患者外周血CD4^＋CD25^＋T细胞及Foxp3mRNA表达的变化

目的通过比较Graves病患者和正常人外周血中CD4^＋和CD8^＋T细胞占淋巴细胞、CD4^＋CD25^＋T细胞占CD4^＋T细胞的比例以及相关基因Foxp3mRNA表达的变化,探讨Graves病患者免疫机制中CD4^＋CD25^＋T细胞亚群所起的作用。方法选择40例初次确诊Graves病的患者和40名健康自愿者,通过流式细胞仪检测外周血CD4^＋CD8^＋T细胞占淋巴细胞、CD4^＋CD25^和CD4^＋CD25^high细胞占CD4^＋T细胞的比例以及CD4^＋CD25^＋T细胞的荧光强度。应用Real—Time PCR检测外周血单个核细胞Foxp3mRNA的表达。结果Graves病患者外周血中CD4^＋T细胞比例以及CD4^＋／CD8^＋的比值明显升高,而CD4^＋CD25^和CD4^＋CD25^highT细胞占CD4^＋T细胞的比例以及CD4^＋CD25^＋T细胞的荧光强度与正常人比较差异无统计学意义,Graves病患者外周血单个核细胞Foxp3mRNA的表达也无明显减少。结论Graves病主要通过效应性CD4^＋T细胞的体液免疫反应对甲状腺组织产生影响,CD4^＋CD25^＋T细胞的免疫抑制作用减弱可能仅是Graves病发病机制中的一个次要环节。     

Suppression of myasthenogenic responses of a T cell line by a dual altered peptide ligand by induction of CD4+CD25+ regulatory cells

Myasthenia gravis is a T cell-dependent, antibody-mediated autoimmune disease. A dual altered peptide ligand (APL) that is composed of the tandemly arranged two single amino acid analogs of two myasthenogenic peptides, p195-212 and p259-271, was demonstrated to down-regulate in vitro and in vivo myasthenia gravis-associated autoreactive responses. The aims of this study were to demonstrate the suppressive properties and to elucidate the mechanism of action of the dual APL on a T cell line specific to the myasthenogenic peptide p195-212. We demonstrate here that incubation of cells of the line with the dual APL resulted in the inhibition of proliferation and secretion of IL-2 and IFN-gamma triggered by p195-212. In contrast, secretion of TGF-beta and IL-10 was upregulated. The dual APL induced the generation of CD4+CD25+ cells that were characterized by the expression of CD45Rb(low), cytotoxic T lymphocyte-associated antigen-4, TGF-beta, CD62L, Foxp3, and neuropilin. In addition, the dual APL-treated cells were capable of inhibiting the proliferation response of the line when the two sets of cells were cocultured. The role of CD4+CD25+ cells was further confirmed by demonstrating that the suppression was abrogated by blocking/neutralization of CD25. Thus, the dual APL acts by inducing the formation of CD4+CD25+ regulatory cells. By using a T cell line, we could show that the immunosuppressive CD4+CD25+ cells were indeed induced by the dual APL and are not part of the naturally occurring regulatory cells.     

结核性和癌性胸腔积液中白细胞介素-16的测定及其意义

目的通过检测结核性和癌性胸腔积液中IL-16水平,探讨其与T淋巴细胞亚群之间的关系。方法收集62例胸腔积液（32例结核性和30例癌性）患者的胸腔积液与静脉血,ELISA测胸腔积液上清液及血清中IL-16水平;流式细胞仪测胸腔积液中T淋巴细胞亚群;行胸腔积液中细胞计数及分类。结果62例胸腔积液者,胸腔积液中IL-16水平高于其血清水平。结核性胸腔积液中IL-16水平高于癌性胸腔积液中IL-16水平。胸腔积液中IL-16水平与淋巴细胞总数、T细胞数及CD4^＋细胞数呈正相关（r分别为0．526、0．638、0．701,P值均小于0．001）。结论与癌性胸腔积液相比,结核性胸腔积液中IL-16水平高,IL-16有可能在CD4^＋ T细胞浸润到胸腔的过程中具有重要的趋化作用。     

Regulatory potential and control of Foxp3 expression in newborn CD4+ T cells

Thymectomy at day 3 after birth leads to autoimmune disease in some genetic backgrounds. Disease is thought to be caused by the lack/paucity of regulatory T cells. We show that 3-day-old mice already contain a significant compartment of Foxp3-expressing CD25(+)CD4(+) splenocytes. Whereas, in adult spleen, the subsets of regulatory T cells (CD25(+) and/or CD103(+)) express high amounts of Foxp3 mRNA, in 3-day-old mice, both thymic and splenic CD25(+)CD4(+) T cell subsets express lower amounts of Foxp3 mRNA, and CD103(+) cells are barely detected. In adult day 3-thymectomized mice, the CD25(+)CD4(+) T cell subset is overrepresented (most of the cells being CD103(+)) and expresses high amounts of Foxp3 mRNA, independent of the development of autoimmune gastritis. These cells control inflammatory bowel disease and the homeostatic expansion of lymphocytes. This study demonstrates that the peripheral immune system of newborn mice is endowed of a remarkable regulatory potential, which develops considerably in the absence of thymic supply.     

Intratumoral CD4+CD25+ regulatory T-cell-mediated suppression of infiltrating CD4+ T cells in B-cell non-Hodgkin lymphoma

Most non-Hodgkin lymphomas (NHLs) are of B-cell origin, but the tumor tissue can be variably infiltrated with T cells. In the present study, we have identified a subset of CD4(+)CD25(+) T cells with high levels of CTLA-4 and Foxp3 (intratumoral T(reg) cells) that are overrepresented in biopsy specimens of B-cell NHL (median of 17% in lymphoma biopsies, 12% in inflammatory tonsil, and 6% in tumor-free lymph nodes; P = .001). We found that these CD4(+)CD25(+) T cells suppressed the proliferation and cytokine (IFN-gamma and IL-4) production of infiltrating CD4(+)CD25(-) T cells in response to PHA stimulation. PD-1 was found to be constitutively and exclusively expressed on a subset of infiltrating CD4(+)CD25(-) T cells, and B7-H1 could be induced on intratumoral CD4(+)CD25(+) T cells in B-cell NHL. Anti-B7-H1 antibody or PD-1 fusion protein partly restored the proliferation of infiltrating CD4(+)CD25(-) T cells when cocultured with intratumoral T(reg) cells. Finally, we found that CCL22 secreted by lymphoma B cells is involved in the chemotaxis and migration of intratumoral T(reg) cells that express CCR4, but not CCR8. Taken together, our results suggest that T(reg) cells are highly represented in the area of B-cell NHL and that malignant B cells are involved in the recruitment of these cells into the area of lymphoma.     

Development of autologous cytotoxic CD4+ T clones in a human model of B-cell non-Hodgkin follicular lymphoma

Immunotherapy for cancer aims to generate cytotoxic cells that are capable of eradicating tumour cells. It has been well demonstrated that helper, non-cytotoxic CD4(+) T cells are important for the induction and maintenance of anti-tumour immunity exerted by cytotoxic CD8(+) T cells. In contrast, the existence of direct anti-tumour, effector cytotoxic CD4(+) T cells remains elusive, mainly due to the paucity of reliable experimental data, especially in human B-cell non-Hodgkin lymphomas. This study developed an appropriate, autologous follicular B-cell non-Hodgkin follicular lymphoma model, including the in vitro establishment of a malignant, human leucocyte antigen class I (HLA-I) deficient B-cell line, and the generation of three autologous anti-tumour cytotoxic CD4(+) T-cell clones originating from the peripheral blood of the same patient. These three clones were considered as tumour specific, because they were capable of killing the malignant, HLA-I-deficient B-cell line through a classical HLA-II restricted perforin-mediated pathway, but did not lyse the Epstein-Barr virus-infected autologous normal B lymphocytes. All three CD4(+)clones were T-cell receptor Vbeta17-Dbeta1-Jbeta1.2 and exhibited an identical complementarity-determining region 3, suggesting the immunodominance of a single peptide antigen presented by tumour cells. Such lymphoma models would provide a useful tool for in vivo expansion and the adoptive transfer of selected CD4(+) cytotoxic cells in immunotherapeutic strategies.