哮喘患者外周血Treg和Th1/Th2的变化及其与哮喘病情的关系

目的：探讨哮喘患者外周血调节性T细胞（Treg）以及辅助性T细胞（Th1/Th2）的比例的变化,探讨其在哮喘的临床治疗中的作用。方法：80例哮喘患者（哮喘组）按临床表现分为急性发作期组（54例）和缓解期组（26例）,同时选择50例健康体检者。应用流式细胞仪检测上述各组外周血CD4＋CD25＋Foxp3＋Treg、CD4＋IFN-γ＋Th1和CD4＋IL-4＋Th2细胞水平,并进行统计学分析。结果：哮喘组CD4＋CD25＋Foxp3＋Treg水平亦明显低于正常对照组（P〈0.05。其中急性发作期组Treg水平明显低于缓解期组和正常对照组（P〈0.05）。而哮喘组Th1/Th2比值显著低于对照组（P〈0.05）,且在哮喘急性发作组中Th1/Th2比值显著低于缓解期组和正常对照组（P〈0.05）。结论：提示Treg和Th在哮喘的发生和发展中起着重要的作用。     

哮喘患者外周血Treg和Th1/Th2的变化及其与哮喘病情的关系

目的:探讨哮喘患者外周血调节性T细胞(Treg)以及辅助性T细胞(Th1/Th2)的比例的变化,探讨其在哮喘的临床治疗中的作用。方法:80例哮喘患者(哮喘组)按临床表现分为急性发作期组(54例)和缓解期组(26例),同时选择50例健康体检者。应用流式细胞仪检测上述各组外周血CD4+CD25+Foxp3+Treg、CD4+IFN-γ+Th1和CD4+IL-4+Th2细胞水平,并进行统计学分析。结果:哮喘组CD4+CD25+Foxp3+Treg水平亦明显低于正常对照组(P<0.05。其中急性发作期组Treg水平明显低于缓解期组和正常对照组(P<0.05)。而哮喘组Th1/Th2比值显著低于对照组(P<0.05),且在哮喘急性发作组中Th1/Th2比值显著低于缓解期组和正常对照组(P<0.05)。结论:提示Treg和Th在哮喘的发生和发展中起着重要的作用。     

老年COPD疾病进展与机体调节性T 细胞的关系分析

目的:探讨老年慢性阻塞性肺疾病(COPD)患者疾病进展与机体调节性T细胞(Treg)的关系。方法:选取我院收治的65例COPD患者(COPD组)以及同期在我院行体检的健康人群45例(正常对照组),将COPD患者分为急性期组41例及稳定期组24例,采用肺功能仪检测肺功能,采用酶联免疫吸附法检测血清γ-干扰素(IFN-γ)、白介素4(IL-4)、IL-17水平,采用流式细胞仪检测外周血CD4+CD25+Treg细胞比例。结果:与正常对照组比较,COPD患者的FEV1、FVC、PEF、FEV1/FVC、6MWT、血清IL-4水平、外周血CD4~+CD25~+Treg细胞比例均明显下降,血清IFN-γ、IL-17及Th1/Th2均显著升高。急性期COPD患者的CD4~+CD25~+Treg细胞比例较正常对照组显著升高,而正常对照组患者的CD4~+CD25~+Treg细胞比例较稳定组COPD患者显著升高,组间比较均有明显差异(P0.05)。结论:老年CODP患者体内存在免疫功能失调,调节性T细胞可能参与了老年COPD疾病的发病以及急性加重过程,导致患者出现肺功能改变。     

Dendritic cells reduce number and function of CD4+CD25+ cells in cytokine-induced killer cells derived from patients with pancreatic carcinoma

Aim and background: CD4⁺CD25⁺ cells are described as professional regulatory/suppressor T cells that are crucial for the prevention of spontaneous autoimmune diseases. They play an important role in maintaining a balanced peripheral immune system. On the other hand, it has been suggested that regulatory T cells (Treg) suppress antitumor immune responses after tumor-specific vaccinations. Therefore, we determined the percentage of regulatory T cells in cytokine-induced killer (CIK) cells, an effector cell population with high impact for adoptive immunotherapeutic strategies. Results: CIK cells showed strong induction of CD4⁺CD25⁺ cells with high secretion of interleukin 10 (IL-10) after unspecific stimulation of the TCR complex and stimulation with interleukin 2. Depletion of CD25⁺ cells led to an increase in cytotoxic activity and a reduction of IL-10 release. A more pronounced reversal of suppression could be induced by coculture of CIK cells with dendritic cells (DCs). After coculture of CIK cells with DCs, the number of CD4⁺CD25⁺ cells as well as the IL-10 concentration in the supernatant decreased, and the cytotoxic activity against pancreatic carcinoma cells increased. This was shown for cells from healthy donors as well as for cells from patients with pancreatic carcinoma. Conclusion: Our established effector cells possess some regulatory features induced by unspecific TCR-activation that could be prevented by coculture with DCs. CIK cells have desirable properties for immunotherapeutical approaches, especially after coculture with DCs, which could be used additionally for induction of a specific immune response.     

Regulatory T cells (CD4CD25FoxP3) expansion in systemic sclerosis correlates with disease activity and severity

Background

The role and function of T regulatory (Treg) cells have not been fully investigated in patients with systemic sclerosis (SSc).

Methods

Ten patients with SSc donated 20 ml of peripheral blood. Activity (Valentini) and severity (Medsger) scores for SSc were calculated for all patients. Healthy volunteers (controls) were matched to each patient by gender and age. CD4⁺ cells were separated using the MACS system. The numbers of Treg cells were estimated by flow cytometry after staining for CD4, CD25, and FoxP3 and calculated as patient-to-control ratio separately for each experiment. Correlations with activity and severity indices of the disease were performed. Twenty-four-hour production of TGF-β and IL-10 by activated CD4⁺ cells was measured by ELISA in culture supernatants.

Results

The numbers of Treg cells, expressed as patient-to-control ratio, correlated significantly with both activity and severity indices (r = 0.71, p = 0.034 and r = 0.67, p = 0.044, respectively). ELISA-measured production of TGF-β and IL-10 by CD4⁺ cells was similar in patients and controls.

Conclusions

Increased numbers of Treg cells are present in patients with SSc, correlating with activity and severity of the disease. This expansion of Treg cells was not accompanied, however, by heightened TGF-β or IL-10 production. Further studies to elaborate the causes and functional significance of Treg cell expansion in SSc are needed.     

Changes of frequency and expression level of CD161 in CD8+ T cells and natural killer T cells in peripheral blood of patients with systemic lupus erythematosus

Immunologic abnormalities of natural killer (NK) cells and T cells play a role in the pathogenesis of systemic lupus erythematosus (SLE). CD161 is expressed on most of the NK cells and on some T cells. The quantities of CD161-expressing cells and expression levels of CD161 were analyzed in T cells and NK cells from patients with SLE compared with normal controls. The expression of CD161 on NK cells, NKT cells, CD4⁺ T cells, and CD8⁺ T cells in peripheral blood from patients with inactive SLE and active SLE, and from the normal controls group were determined using flow cytometry. The frequency and expression level of CD161 in the lymphocyte subsets and its relationship with the quantity of regulatory T cells, anti-double stranded DNA antibody, and the titer of antinuclear antibody were evaluated. Both the percentages of the CD161⁺ subpopulation and the mean fluorescence intensities (MFIs) of CD161 in CD8⁺ T cells and NKT cells decreased significantly in SLE patients compared with normal controls (P < .001). The CD161 expression in CD8⁺ T cells and NKT cells also decreased in the anti-dsDNA (+) group (P < .05). The counts of Treg cells were lower in SLE patients and were weakly correlated with the percentage of the CD161 subpopulation (r = 0.229, P = .016) and the MFIs of CD161 expression in CD8⁺ T cells (r = .232, P = .014). The frequencies and levels of CD161 expression on CD8⁺ T cells and NKT cells were reduced in SLE patients, suggesting that an abnormality of these cells was related to the pathogenesis of SLE.     

变应性鼻炎患者外周血中IL-27和Treg细胞的相关性分析

目的探讨外周血IL-27和CD4^＋CD25^＋调节性T细胞（Treg）在变应性鼻炎（AR）发病机制中的作用。方法2012年3月至7月,收集AR患者32例（AR组）和20例健康志愿者（对照组）外周血,采用流式细胞术（FCM）检测外周血中Treg细胞比例;ELISA检测血清中IL-27、IL-10和TGF-β1的水平。结果AR组Treg细胞百分率[（1．75±0．56）％]明显低于对照组[（4．76±1．75）％],两组比较的差异有统计学意义（P〈0．01）。AR组IL-27、IL-10和TGF-β1的水平分别为（24．43±16．36）pg／ml、（14．29±6．16）pg／ml、（0．34±0．04）pg／ml,均低于对照组（44．09±13．12）pg／ml、（31．32±21．20）pg／ml、（O．49±0．06）pg／ml,两组之间的差异有统计学意义（P〈0．01）。AR患者外周血中IL-27和Treg细胞百分率、IL-10、TGF-β1存在正相关（r分别为0．825,0．646,0．517,P〈0．01）,Treg细胞百分率和IL-10、TGF-β1存在正相关（r=0.622,0．738,P〈0．01）,IL-10和TGF-β1无相关性（r=0．304,P〉0．05）结论AR患者外周血中IL-27水平降低,Treg细匏百分率降低及其主要分泌因子IL-10、TGF-β1水平降低,且IL-27与Treg细胞百分率、IL-10、TGF-β1水平呈正相关,提示在AR发病中IL-27对Treg细胞可能具有免疫调节作用。     

Differential expression of interferon-gamma,IL-4 and IL-10 in peripheral blood mononuclear cells during early pregnancy of the bovine

Maternal systemic immune response is regulated by conceptus-derived signals through peripheral blood mononuclear cells (PBMCs) via blood circulation during early pregnancy in cattle. In this study, the PBMCs from day 18 in non-pregnant cows and days 14, 18 and 30 in pregnant cows were used to explore the expression of interferon-gamma (IFN-γ), interleukin 4 (IL-4) and IL-10, and the plasma progesterone (P4) concentration was also determined. The results showed that the expression levels of mRNA and the protein of IFN-γ were lower and that IL-4 and IL-10 were higher in the PBMCs from the pregnant cows than in those of non-pregnant cows. From this study, early pregnancy induced a lower Th1 immunity (IFN-γ) and a higher Th2 immunity (IL-4 and IL-10) in the PBMCs, which may be related to interferon-tau and P4, thereby contributing to successful pregnancy in cattle.     

Differential expression of bitter taste receptors in non-cancerous breast epithelial and breast cancer cells

The human bitter taste receptors (T2Rs) are chemosensory receptors that belong to the G protein-coupled receptor superfamily. T2Rs are present on the surface of oral and many extra-oral cells. In humans 25 T2Rs are present, and these are activated by hundreds of chemical molecules of diverse structure. Previous studies have shown that many bitter compounds including chloroquine, quinidine, bitter melon extract and cucurbitacins B and E inhibit tumor growth and induce apoptosis in cancer cells. However, the existence of T2Rs in cancer cell is not yet elucidated. In this report using quantitative (q)-PCR and flow cytometry, we characterized the expression of T2R1, T2R4, T2R10, T2R38 and T2R49 in the highly metastatic breast cancer cell line MDA-MB-231, poorly metastatic cell line MCF-7, and non-cancerous mammary epithelial cell line MCF-10A. Among the 5 T2Rs analyzed by qPCR and flow cytometry, T2R4 is expressed at 40–70% in mammary epithelial cells in comparison to commonly used breast cancer marker proteins, estrogen receptor and E-cadherin. Interestingly, the expression of T2R4 was downregulated in breast cancer cells. An increase in intracellular calcium mobilization was observed after the application of bitter agonists, quinine, dextromethorphan, and phenylthiocarbamide that are specific for some of the 5 T2Rs. This suggests that the endogenous T2Rs expressed in these cells are functional. Taken together, our novel findings suggest that T2Rs are differentially expressed in mammary epithelial cells, with some T2Rs downregulated in breast cancer cells.     

Uptake of donor lymphocytes treated with 8-methoxypsoralen and ultraviolet A light by recipient dendritic cells induces CD4CD25Foxp3 regulatory T cells and down-regulates cardiac allograft rejection

Extracorporeal photopheresis (ECP) is an effective immunomodulatory therapy and has been demonstrated to be beneficial for graft-vs-host disease and solid-organ allograft rejection. ECP involves reinfusion of a patient’s autologous peripheral blood leukocytes treated ex vivo with 8-methoxypsoralen and UVA light radiation (PUVA). Previous studies focused only on ECP treatment of recipient immune cells. Our study is the first to extend the target of ECP treatment to donor immune cells. The results of in vitro co-culture experiments demonstrate uptake of donor PUVA-treated splenic lymphocytes (PUVA-SPs) by recipient immature dendritic cells (DCs). Phagocytosis of donor PUVA-SPs does not stimulate phenotype maturation of recipient DCs. In the same co-culture system, donor PUVA-SPs enhanced production of interleukin-10 and interferon-γ by recipient DCs and impaired the subsequent capability of recipient DCs to stimulate recipient naïve T cells. Phagocytosis of donor PUVA-SP (PUVA-SP DCs) by recipient DCs shifted T-cell responses in favor of T helper 2 cells. Infusion of PUVA-SP DCs inhibited cardiac allograft rejection in an antigen-specific manner and induced CD4⁺CD25^highFoxp3⁺ regulatory T cells. In conclusion, PUVA-SP DCs simultaneously deliver the donor antigen and the regulatory signal to the transplant recipient, and thus can be used to develop a novel DC vaccine for negative immune regulation and immune tolerance induction.     

支气管哮喘患者呼吸道菌群及FeNO、Th/Treg细胞水平变化

探究支气管哮喘患者呼吸道菌群及呼出气一氧化氮(FeNO)、Th17细胞与调节性T细胞(Treg)水平变化。

选取2017年5月至2019年8月我院呼吸内科收治的60例支气管哮喘患者为观察组, 选择同期35例健康者作为对照组。观察两组对象呼吸道菌群, FeNO、Th17细胞、Treg细胞, T细胞亚群及免疫球蛋白水平。

观察组患者共分离出87株细菌, 其中奈瑟菌检出率最高, 其次为肺炎链球菌、甲型链球菌、表皮葡萄球菌、韦荣球菌; 对照组对象共分离出35株细菌, 其中甲型链球菌检出率最高, 其次为表皮葡萄球菌、奈瑟菌、韦荣球菌、消化球菌。观察组患者甲型链球菌占比低于对照组(χ²=4.554, P=0.032), 其余菌群比较差异无统计学意义(均P > 0.05)。观察组患者FeNO、Th17细胞、Treg细胞水平及Th17/Treg均高于对照组, 差异有统计学意义(均P < 0.05)。观察组患者CD4⁺细胞、CD4⁺/CD8⁺低于对照组, 差异有统计学意义(均P < 0.05);而两组对象CD3⁺、CD8⁺细胞水平比较差异无统计学意义(均P > 0.05)。观察组患者免疫球蛋白G(IgG)、免疫球蛋白A(IgA)、免疫球蛋白M(IgM)水平与对照组比较差异无统计学意义(均P > 0.05)。

支气管哮喘患者呼吸道检出率较高的菌群为奈瑟菌, 且FeNO、Th17/Treg水平均较高, 细胞免疫功能低下, 应及时进行治疗。

     

Vitamin E, signalosomes and gene expression in T cells

CD4+T cells from aged humans or mice show significant reductions in IL-2 production upon activation. The resulting decreased proliferation is linked to higher risks of infection in the elderly. Several lines of evidence indicate that intrinsic defects preferentially affecting the naïve subset of CD4+T cells contribute to this reduced IL-2 production. Comparison of the biochemical pathways that transduce activation signals from the T cell receptor to the IL-2 promoter in young and old CD4+T cells has demonstrated age-related impairments at initial molecular events, in particular the phosphorylation of kinases and adapter proteins involved in the formation of signalosomes - complex multiprotein assemblies that provide the framework for effective signal transduction. Confocal microscopy has demonstrated a series of age-related impairments in effective immune synapse formation. Vitamin E can reverse many of these CD4+T cell age-associated defects, including reduced levels of phosphorylation of critical signaling/adapter proteins as well as defective immune synapse formation. Vitamin E also enhances IL-2 production, expression of several cell cycle control proteins, and proliferation. Although the precise mechanisms underlying this effect are not understood, it is possible that this antioxidant lipophilic vitamin can prevent the propagation of polyunsaturated fatty acid peroxidation in the cell membrane, influence the biochemical characteristics of specific lipid bilayer microdomains involved in signal transduction, modulate the activity of kinases/phosphatases, or interact with intracellular receptors.     

Immune responses to p53 in patients with cancer:enrichment in tetramer+ p53 peptide-specific T cells and regulatory T cells at tumor sites

Objective: A majority of human cancers, including head and neck cancer (HNC), overexpress p53. Although T cells specific for wild-type (wt) sequence p53 peptides are detectable in the peripheral blood of patients with HNC, it is unknown whether such T cells accumulate in tumor-involved tissues. Also, the localization of regulatory T cells (Treg) to tumor sites in HNC has not been investigated to date. Methods: Tumor infiltrating lymphocytes (TIL), tumor-involved or non-involved lymph node lymphocytes (LNL) and peripheral blood mononuclear cells (PBMC) were obtained from 24 HLA-A2.1+ patients with HNC. Using tetramers and four-color flow cytometry, the frequency of Treg and CD3+CD8+ T cells specific for wt p53 epitopes as well as their functional attributes were determined. Results: The CD3+CD8+ tetramer+ cell frequency was significantly higher (P<0.001) in TIL than autologous PBMC as was the percentage of CD4+CD25+ T cells (P<0.003). TIL were enriched in FOXp3+, GITR+ and CTLA-4+ Treg. CD8+ TIL had low expression and produced little IFN- after ex vivo stimulation relative to autologous PBMC or PBMC from NC. Conclusions: Anti-wt p53 epitope-specific T cells and Treg preferentially localize to tumor sites in patients with HNC. However, despite enrichment in tumor peptide-specific T cells, the effector cell population (CD3+CD8+) in TIL or PBMC was unresponsive to activation in the tumor microenvironment enriched in Treg.     

Inducible gene expression with the Tet-on system in CD4+ T cells and thymocytes of mice

CD4+ T cells with their growing list of effector and regulatory subpopulations have vital functions within the immunohematopoietic system. We report here on the first mouse lines that allow temporally and quantitatively controlled expression of transgenes specifically in CD4+ thymocytes and T cells. These were constructed using the Tet-on system. The rtTA2(S)-M2 version of the reverse tetracycline-dependent transactivator was placed under control of all known CD4 regulatory elements. Reporter transgene expression in mice expressing these constructs is highly specific for CD4+ cells, is strictly dependent on the tetracycline derivative doxycycline, and can be regulated by up to five logs depending on the doxycycline concentration. Moreover, we demonstrate that these mice can be used for noninvasive in vivo imaging of a coexpressed luciferase reporter. These new mouse lines should be highly valuable for studying and manipulating numerous aspects of CD4+ T cell development, biology, and function.     

Gene expression profiles in human peripheral blood mononuclear cells as biomarkers for nutritional in vitro and in vivo investigations

Identification of chemopreventive substances may be achieved by measuring biological endpoints in human cells in vitro. Since generally only tumour cells are available for such investigations, our aim was to test the applicability of peripheral blood mononuclear cells (PBMC) as an in vitro primary cell model since they mimic the human in vivo situation and are relatively easily available. Cell culture conditions were refined, and the basal variation of gene expression related to drug metabolism and stress response was determined. Results were compared with profiles of an established human colon cell line (HT29) as standard. For biomarker development of nutritional effects, PBMC and HT29 cells were treated with potentially chemopreventive substances (chrysin and butyrate), and gene expression was determined. Key results were that relevant stress response genes, such as glutathione S-transferase T2 (GSTT2) and GSTM2, were modulated by butyrate in PBMC as in HT29 cells, but the blood cells were less sensitive and responded with high individual differences. We conclude that these cells may serve as a surrogate tissue in dietary investigations and the identified differentially expressed genes have the potential to become marker genes for population studies on biological effects.     

Systemic immune effects of adjuvant chemotherapy with 5-fluorouracil,epirubicin and cyclophosphamide and/or radiotherapy in breast cancer: a longitudinal study

Immunotherapy is being increasingly utilized for adjuvant treatment for breast cancer (BC). We have previously described immune functions during primary therapy for BC. The present study describes immune recovery patterns during long-term, unmaintained follow-up after completion of adjuvant therapy.A group of patients with primary BC had been treated with adjuvant radio-chemotherapy (RT + CT) 5-fluorouracil, epirubicin and cyclophosphamide (FEC) (n = 21) and another group with radiotherapy (RT) (n = 20) alone. Immunological testing of NK and T-cell functions was performed initially at the end of adjuvant treatment and repeated after 2, 6 and 12 months. NK cell cytotoxicity was significantly higher (P < 0.05) at all time-points in patients than in age-matched controls and did not differ between the two treatments groups during one year observation. In contrast, lower numbers of CD4 T-cells and lower expression of CD28 on T-cells was observed particularly in RT + CT patients and did not normalize during the observation period. The numbers of T_reg cells (CD4⁺CD25^high) were low in the RT + CT group during follow-up, as well as expression of TCRξ, Zap70, p56^lck, P59^fyn and PI3 k in CD4⁺ cells. In contrast, expression of intracellular cytokines (IFN-γ, IL-2, IL-4) in CD4 and CD8 T cells were significantly higher in RT + CT patients than in the RT group and the difference increased during follow-up. In conclusion, NK-cell cytotoxicity increased during unmaintained long-term follow-up whereas CD4 and regulatory T cells as well as signal transduction molecules remained low following adjuvant radio-chemotherapy.     

Genomic profiles for human peripheral blood T cells, B cells, natural killer cells, monocytes, and polymorphonuclear cells: comparisons to ischemic stroke, migraine, and Tourette syndrome

Blood genomic profiling has been applied to disorders of the blood and various organ systems including brain to elucidate disease mechanisms and identify surrogate disease markers. Since most studies have not examined specific cell types, we performed a preliminary genomic survey of major blood cell types from normal individuals using microarrays. CD4+ T cells, CD8+ T cells, CD19+ B cells, CD56+ natural killer cells, and CD14+ monocytes were negatively selected using the RosetteSep antibody cocktail, while polymorphonuclear leukocytes were separated with density gradient media. Genes differentially expressed by each cell type were identified. To demonstrate the potential use of such cell subtype-specific genomic expression data, a number of the major genes previously reported to be regulated in ischemic stroke, migraine, and Tourette syndrome are shown to be associated with distinct cell populations in blood. These specific gene expression, cell-type-related profiles will need to be confirmed in larger data sets and could be used to study these and many other neurological diseases.