Patterns of A2A extracellular adenosine receptor expression in different functional subsets of human peripheral T cells. Flow cytometry studies with anti-A2A receptor monoclonal antibodies

Signaling through A2A adenosine receptors (A2AR) regulates T lymphocyte expansion and modulates T cell receptor (TCR)-mediated effector functions in vitro. To understand the role of A2ARs in the regulation of immune response, we investigated the expression levels of this receptor in different functional lymphocyte subsets. Monoclonal anti-A2AR antibody was used to develop a flow cytometric assay to quantify the expression A2ARs on lymphocytes. We report that detectable levels of expression of A2ARs are much higher among T cells than B cells. More CD4(+) than CD8(+) T cells express A2ARs, but activation of T cells increases A2AR expression, predominantly in CD8(+) T cells. No significant differences were found in the proportion of A2AR+ cells between CD8(low) and CD8(high) T cells or between TCR/CD3(low) and TCR/CD3(high) T cells. Studies of T helper cell subsets (TH1 and TH2) reveal that lymphokine-producing cells are much more likely to express A2ARs than are cells that do not produce lymphokines. These results suggest that A2ARs are variably expressed on T cell subsets and may regulate cytokine production in activated T lymphocytes.     

Quantitation of immunosuppression by tacrolimus using flow cytometric analysis of interleukin-2 and interferon-gamma inhibition in CD8(-) and CD8(+) peripheral blood T cells

The authors have determined the frequency of intracellular interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) synthesis by T-cell subsets in whole blood (WB) and isolated lymphocytes in 16 transplant recipients treated with tacrolimus and 10 control patients who were not transplant recipients. The authors also determined the impact of varying amounts of red blood cells (RBC) on immunosuppression by tacrolimus. Samples were analyzed by two-color flow cytometry, and the results were expressed as a ratio of whole blood to isolated lymphocytes. In healthy subjects who were not transplant recipients, the frequency of IL-2--producing CD8(-) and CD8(+) cells was higher in WB than in isolated lymphocytes (mean +/- SD of whole blood to lymphocytes ratio: 1.24 +/- 0.5 and 1.67 +/- 0.62, respectively). Adding varying amounts of RBC had no significant impact on IL-2 production by CD8(-) and CD8(+) T cells. Adding tacrolimus (10 ng/mL) to lymphocyte cultures inhibited (90%) IL-2 production in isolated T cells but not in the whole-blood assay. The dose of tacrolimus required for a 50% inhibition of IL-2 release in T cells was 10-fold higher in cultures with RBC than without. Peripheral blood mononuclear cells (PBMC) isolated from tacrolimus-treated whole blood (WB) showed less IL-2 inhibition than did lymphocytes in the WB. The authors also tested cytokine production in WB and PBMCs in 16 transplant recipients and observed various patterns of reactivity. The frequency of IL-2--producing CD8(-) and CD8(+) cells was similar using two different methods in 10 of 16 patients tested. By contrast, in the remaining six patients the authors observed a significant inhibition of IL-2 production in both CD8(-) and CD8(+) T-cell subsets in the whole-blood assay but not in the isolated lymphocytes. The frequency of CD8(-) IFN-gamma--producing cells was significantly lower in 9 of 16 patients, but the same individuals showed no inhibition of their CD8(+) IFN-gamma T cells. The trough levels of tacrolimus did not predict the level of cytokine inhibition in the whole-blood assay in these patients. The authors' results show that the whole-blood assay for cytokine production can be used for monitoring the in vivo effect of tacrolimus in transplant recipients.     

Cardiotoxin-III selectively enhances activation-induced apoptosis of human CD8+ T lymphocytes

Cardiotoxin-III (CTX-III), a major cardiotoxin isolated from the venom of the Taiwan cobra (Naja naja atra), is a highly basic, hydrophobic, toxic protein, which can induce lysis of mononuclear cells by an unknown mechanism. This study was undertaken to investigate the effects of CTX-III on untreated and PHA-activated peripheral blood mononuclear cells (PBMCs) in vitro. The results show that treatment of PHA-activated lymphocytes with CTX-III (10 microg/ml) induced apoptosis and depletion of the CD8(+) population. In both untreated and PHA-treated lymphocytes, interferon-gamma production was dramatically reduced and interleukin-2 (IL-2) production was moderately reduced by CTX-III treatment. In PHA-activated lymphocytes, CD4 expression was increased, whereas CD8 and IL-2R beta chain (CD25) expression were decreased. In contrast, CTX-III had no effect on the viability of PHA-activated monocytes but significantly enhanced their tumor necrosis factor-alpha production. These results show that CTX-III selectively enhanced activation-induced apoptosis in CD8(+) T cells. CTX-III was found to bind to the cell membrane of PHA-stimulated PBMCs, and three CTX-III-binding proteins, with molecular weights of 92, 77, and 68 kDa, were identified. We therefore propose that CTX-III interacts with one or more cell surface proteins and initiates a signal pathway causing functional changes. These findings provide an insight into the immunomodulatory properties of CTX-III and suggest a novel method for the selective induction of apoptosis in CD8(+) T lymphocytes.     

Differential induction of glucocorticoid-dependent apoptosis in murine lymphoid subpopulations in vivo following coexposure to lipopolysaccharide and vomitoxin (deoxynivalenol)

Lipopolysaccharide (LPS) and vomitoxin (VT) synergistically induce glucocorticoid- mediated apoptotic cell death in lymphoid tissues of the mouse. Based on the known effects of glucocorticoids, it was hypothesized that the combined exposure to LPS and VT targets immature lymphocyte populations. To test this hypothesis, we quantified the effects of VT and LPS on apoptosis induction in T lymphocyte subsets in thymus and B lymphocyte subsets in Peyer's patches and bone marrow. Flow cytometry revealed that a single dose of LPS (0.1 mg/kg body wt ip) together with VT (12.5 mg/kg body wt po) promoted apoptosis of immature (CD4(-)CD8(-), CD4(+)CD8(+)) and mature (CD4(-)CD8(+)) thymocytes at 12 h with a subsequent reduction of these populations being detectable at 24 h. RU 486, a glucocorticoid receptor antagonist, significantly abrogated apoptosis in CD4(-)CD8(-), CD4(+)CD8(+), and CD4(-)CD8(+) subsets and also prevented loss in cell numbers. In Peyer's patches, mature-B lymphocytes (B220(+)IgM(-)IgD(+)) underwent apoptosis and, in bone marrow, pro/pre-B lymphocytes (B220(+)IgM(-)IgD(-)) and mature-B lymphocytes (B220(+)IgM(-)IgD(+)) underwent apoptosis at 12 h after toxin co- exposure. RU 486 blocked LPS + VT-induced apoptosis of the aforementioned subsets in Peyer patches and bone marrow at 12 h. Taken together, these data suggest that LPS can interact with VT in mice to induce the glucocorticoid-driven apoptotic loss of immature thymocytes and cytotoxic T lymphocytes in thymus, mature-B lymphocytes in Peyer's patch, and pro/pre-B lymphocytes and mature-B lymphocytes in bone marrow in mice.     

CD59在艾滋病感染者CD4^＋T细胞表达及凋亡的影响

目的探讨补体调节蛋白CD59在艾滋病（HIV）感染者外周血CD4^＋T细胞上的表达及与凋亡之间的关系。方法收集12例确诊HIV感染者外周血标本（观察组）,同时收集10例健康对照者外周血标本（对照组）。分离外周血单个核细胞（PBMC）,并进行细胞表面染色。使用BDFACSCanto流式仪检测各项指标,采用FACSDiva软件分析CD4^＋T细胞CD59的表达情况,并分析CD59^＋CD4^＋T、CD59^-CD4^＋T细胞的凋亡情况。结果观察组CD4^＋T细胞CD59表达明显高于对照组（t=5．198,P〈0．01）;CD59＋CD4^＋细胞凋亡比率明显升高（t=5．968,P〈0．01）;而CD59^-CD4^＋T细胞的凋亡比例二组间差异无统计学意义（t=0．1353,P=0．8577）。结论HIV感染可引起CD4^＋T细胞补体调节蛋白CD59的表达,而CD59的表达会使CD4^＋T细胞凋亡增加。     

雷帕霉素诱导Balb/c小鼠CD4~+ CD25~+ foxp3~+调节性T细胞增殖

目的探讨雷帕霉素对Balb/c小鼠CD4+ CD25+ foxp3+调节性T细胞的作用。方法取8wk龄的SPF级Balb/c小鼠30只,随机分为两组,实验组每只灌胃雷帕霉素0.4mg.d-1,对照组灌胃每天予等体积无菌水,共3wk。无菌条件下肝素抗凝心脏采血,分离脾脏,制备单细胞悬液。采用流式细胞仪检测小鼠外周血和脾细胞CD4+CD25+T细胞,实时定量PCR检测小鼠脾细胞foxp3 mRNA的表达。结果实验组小鼠外周血和脾细胞中CD4+CD25+T细胞的比例分别为(9.95±4.65)%和(24.13±10.06)%,对照组小鼠外周血和脾细胞中CD4+CD25+T细胞的比例分别为(5.01±1.49)%和(8.48±3.19)%,差异均有显著性(P<0.01)。实验组小鼠脾细胞foxp3 mRNA的表达水平明显高于对照组,是对照组的6.029倍,差异有显著性(P<0.01)。结论雷帕霉素能够明显诱导Balb/c小鼠体内CD4+CD25+T细胞的增殖,并能提高foxp3+ mRNA的表达。     

造血移植物中CD3~+CD8~(low)和CD3~+CD4~-CD8~(low)细胞亚群的检测及分析

目的检测造血移植物(正常人骨髓、脐血及动员后外周血)中CD3+CD8low和CD3+CD4-CD8low细胞亚群,探讨其促进造血干/祖细胞植入异基因骨髓的功能,以期拓宽脐血移植的应用范围。方法直接三色免疫荧光标记流式细胞术检测。结果CD3+CD8low和CD3+CD4-CD8low细胞亚群占CD3+细胞亚群的比例在骨髓中最高,为(8.61±1.40)%,动员后外周血次之,为(5.11±0.76)%,脐血最低,为(3.31±0.88)%(P<0.01);"初始"T(CD45RA+CD45RO-)细胞亚群占CD8low细胞亚群的比例为脐血(94.26±2.46),骨髓(58.68±7.57),动员后外周血(73.21±3.60),"初始"T占CD8high细胞亚群的比例为脐血(82.63±3.16),骨髓(38.69±3.24),动员后外周血(51.58±4.23),各移植物中"初始"T细胞亚群占CD8low细胞亚群的比例均高于其占CD8high细胞亚群的比例(P<0.01),CD8low细胞亚群中"初始"T与"记忆"T(CD45RA-CD45RO+)细胞亚群的比例均高于二者在CD8high细胞亚群的比例。结论脐血CD8low和CD8lowCD4-细胞占CD3+细胞的比例明显低于骨髓,可能是脐血移植植入延迟的原因之一;"初始T与"记忆"T细胞亚群的比例增高,可能与CD3+CD8low细胞亚群维持和诱导免疫耐受,不引起移植物抗宿主病有关。     

小儿急性白血病患者血清VEGF水平和外周血B细胞及T淋巴细胞亚群水平检测的临床意义

目的探讨小儿急性白血病患者血清血管内皮生长因子（VEGF）水平和外周血B和T淋巴细胞亚群水平及临床意义。方法应用酶联免疫法（ELISA）和单克隆抗体法对31例小儿急性白血病患者进行了血清VEGF和外周B和T淋巴细胞亚群水平的检测，并与30名正常健康儿童作比较。结果小儿急性白血病患者血清VEGF、B淋巴细胞数均显著地高于正常人组（P〈0．01），而CD3、CIM、CIM／CD8比值则显著地低于正常人组（P〈0．01）。结论检测血清VEGF和外周血B细胞和T淋巴细胞亚群水平的变化对观察病情和预后均有十分重要的临床价值。     

猪囊尾蚴病患者CD4~+ CD25~+调节性T细胞水平的变化

目的检测猪囊尾蚴病患者外周血中CD4+CD25+调节性T淋巴细胞及其FOXP3的表达情况,探讨CD4+CD25+调节性T淋巴细胞在猪囊尾蚴感染中的免疫调控作用及意义。方法采用流式细胞仪检测11例猪囊尾蚴病患者外周血中CD4+CD25+T淋巴细胞的含量,同时观察CD4+CD25+T细胞中表达FOXP3群体的百分含量。结果囊尾蚴病患者外周血中CD4+CD25+T淋巴细胞的百分含量为6.11%,较正常人(3.94%)明显升高(P<0.05);患者外周血中CD4+CD25+T细胞表达FOXP3的细胞百分含量为15.67%,与正常对照组(11.09%)有显著性差异(P<0.05)。结论猪囊尾蚴病患者外周血中CD4+CD25+T淋巴细胞的百分含量显著升高,表明CD4+CD25+调节性T细胞可能参与猪囊尾蚴感染的免疫抑制。     

Measurement of circulating T cell subsets positive for Fas antigen (CD95) in patients with alcoholic hepatitis.

T cell subsets positive for Fas antigen in peripheral blood of patients with alcoholic hepatitis were measured, using monoclonal antibodies in two colour immunofluorescence assay with flowcytometry. 1) In the patients with alcoholic hepatitis, the ratio of mean +/- standard deviation (M +/- SD) of CD4+ cells positive for CD95 (Fas antigen) in peripheral blood lymphocytes of patients with alcoholic hepatitis tended to increase, but the ratio of CD95-positive cells in CD4+ cells of peripheral blood was almost the same, compared with those of healthy controls. In the alcoholics (overdrink) who did not show alcoholic hepatitis with or without apparent alcoholic damage, the ratio of CD95-positive CD4+ cells in peripheral blood lymphocytes was within normal range, while CD95-positive cells in CD4+ cells of peripheral blood tended to decrease. 2) In the alcoholic hepatitis, the ratios of CD8+ cells positive for CD95 and CD95-positive cells in CD8+ cells of peripheral blood decreased significantly, and in the alcoholics (overdrink) they also tended to decrease. 3) The fluorescence intensity of CD95 on CD4+ cells in peripheral blood of the alcoholics (overdrink) decreased apparently, although the one on CD8+ cells did not. 4) The ratios of T cell subsets, that is, CD4+ and CD8+ cells, positive for HLADR in peripheral blood of the patients with alcoholic hepatitis increased significantly, respectively. These results showed that the ratio of CD8+ cells positive for Fas antigen in peripheral blood of the patients with alcoholic hepatitis decreased. It was suspected that this finding might be due to the direct effect of intaken alcohol to T cell subsets rather than hepatitis, and/or the result of immunological homeostasis in alcoholic hepatitis.     

多发性骨髓瘤患者外周血CD4~+T淋巴细胞亚群特点研究

目的研究多发性骨髓瘤(MM)患者外周血CD4+T淋巴细胞亚群特点。方法用流式细胞术检测30例MM初诊患者、20例治疗后患者及10名健康志愿者(对照组)外周血CD4+T淋巴细胞亚群,比较分析3组之间调节性T细胞(Treg)、辅助性T细胞(Th)1、Th17比例及Treg/Th17、Th1/Th17比值。结果初诊MM患者外周血Th17比例明显高于对照组,Treg、Th1比例及Treg/Th17、Th1/Th17比值明显低于对照组。治疗后患者Treg、Th1比例及Treg/Th17、Th1/Th17比值较治疗前明显提高,Th1及CD4+CD25+细胞比例基本恢复至对照组水平,CD4+Foxp3+细胞比例及Treg/Th17、Th1/Th17比值仍明显低于对照组。结论 MM患者外周血Th17细胞在初始T细胞的分化过程中取得了极化优势。治疗后Th17细胞极化优势现象明显改善,Th17细胞可能参与了MM的疾病进程。     

Trichloroethylene enhances TCR-CD3-induced proliferation of CD8(+) rather than CD4(+) T cells

Trichloroethylene (TCE) is suspected as a potent immunomodulator that accelerates the development of allergic diseases. We previously reported that TCE promotes ovalbumin (OVA)-induced active cutaneous anaphylaxis, including enhancing antigen-specific serum IgE levels and splenic lymphocyte proliferation. However, the target cells and molecular mechanism through which TCE modulates antigen-specific immune responses remain unclear. To identify a potential underlying mechanism, we investigated whether TCE modulates T cell receptor (TCR)-induced T cell activation and proliferation in vitro. TCE enhanced T cell proliferation primed by anti-CD3 antibody, but not concanavalin A, in a dose-dependent fashion. In addition, TCE enhanced anti-CD3-primed proliferation of CD8(+) rather than CD4(+) T cells. Consistent with this result, TCE markedly enhanced the Lck phosphorylation mediated by anti-CD3 antibody in CD8(+) but not CD4(+) T cells. Furthermore, we analyzed the effect of TCE exposure via drinking water for 2 weeks on splenocyte populations in non-immunized and OVA-immunized mice. In OVA-immunized mice, TCE (3 mg/l) significantly expanded CD3(+), CD8(+) and CD4(+) cell populations, however the effect at the lower concentration was significant only in the CD8(+) populations, whereas TCE had no effect on these cells population in non-immunized mice. These findings suggest that TCE enhances TCR-CD3-induced proliferation of CD8(+) rather than CD4(+) cells and disrupts various activities of peripheral T cells.     

Immune reconstitution after autologous hematopoietic transplantation with Lin-, CD34+, Thy-1lo selected or intact stem cell products

In sequential studies, we compared immune reconstitution following high-dose chemotherapy (HDT) and stem cell transplantation (SCT) using intact mobilized peripheral blood stem cell (PSC) in intermediate grade non-Hodgkin's lymphoma (NHL) patients and CD34(+), lineage-negative (Lin(-)), Thy-1(lo) (CD34(+)Lin(-)Thy-1(lo)) stem cells in low-grade NHL patients. Cytokine expression and cellular phenotype and function were used as the basis of comparison. Despite differences in cellular composition of the stem cell grafts, immune reconstitution in both groups was similar. Significantly higher levels of type 1- and 2-associated cytokine messenger ribonucleic acid (mRNA) were observed both prior to and following transplant in the peripheral blood (PB) of both cohorts as compared to normal individuals. Similar levels of interleukin (IL)-4, IL-10, interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha) messenger ribonucleic acid (mRNA) were seen in PB mononuclear cells following transplant with either product. In contrast, patients receiving isolated CD34(+)Lin(-)Thy-1(lo) cells expressed significantly higher IL-2 levels at all times examined post-transplant. Despite the high levels of cytokine gene expression and rapid restoration to pretransplant levels of CD3 cell number by day 30, T cell function and CD4:CD8 and CD4(+)CD45RA:CD4(+)CD45RO(+) ratios were significantly depressed in both cohorts compared to normal donors, and significantly lower in patients transplanted with CD34(+)Lin(-)Thy-1(lo) compared to patients receiving an intact PSC product. These data suggest that the peripheral tolerance in patients receiving HDT and an autologous SCT occurs independent of graft composition, although immune function and CD4 recovery are better facilitated by transplantation of an intact product.     

急性乙型肝炎外周血淋巴细胞亚群动态分析

目的 探讨急性乙型肝炎(AHB)患者外周血免疫细胞包括T淋巴细胞亚群、B淋巴细胞和自然杀伤(NK)细胞在疾病发展中的动态变化及其意义.方法 流式细胞技术检测17例AHB患者(AHB组)外周血淋巴细胞亚群动态变化和36例慢性乙型肝炎(CHB组)患者及32例正常对照组淋巴细胞亚群分布特点;动态检测AHB患者CD4~+/CD8~+变化,并探讨其与ALT改变及与HBVDNA清除的相关性.结果 AHB组人院4周内外周血CD3~+、CD4~+和CD8~+T细胞频率显著高于CHB组和对照组,NK细胞4周内均低于其余2组(P<0.05).AHB疾病早期ALT高水平异常时CD4~+/CD8~+明显低下;随着ALT恢复正常,CD4~+/CD8~+比值呈逐渐升高趋势.结论 AHB患者外周血免疫细胞的分布与对照组和CHB患者不同,淋巴细胞亚群的动态变化与疾病发展可能有一定的相关性.     

In-vitro generation and characterisation of murine CD4+CD25+ regulatory T cells with indirect allospecificity

Naturally arising CD4(+)CD25(+) regulatory T cells play a pivotal role in the prevention of autoimmunity and in the induction of donor-specific transplantation tolerance. Harnessing regulatory cells for potential adoptive cell therapy is hampered by their lack of antigen-specificity and their limited numbers. Here we describe the generation and expansion of murine CD4(+)CD25(+) T cells with antigen-specificity for an K(d) peptide as potential reagents for adoptive cell therapy in promoting donor-specific transplantation tolerance. Using bone marrow-derived autologous dendritic cells pulsed with the K(d) peptide, we generated T cell lines from purified CD4(+)CD25(+) T cells from C56BL/6 mice. The T cell lines expressed high level of CD25 and low level of CD45RB and CD69. They maintained the expression of CD62L, GITR, CTLA-4 and more importantly FoxP3. The CD4(+)CD25(+) T cell lines were anergic after TCR stimulation and produced little cytokine such as IL-2 and IFN-gamma. Importantly, they were more potent than freshly isolated CD4(+)CD25(+) T cells in suppressing proliferation and cytokine secretion by effector CD4(+) T cells. Furthermore, the CD4(+)CD25(+) T cell lines could be expanded to large cell numbers and maintained in culture up to 1 year. The K(d)-specific CD4(+)CD25(+) T cell lines will be invaluable in devising a strategy for the induction of cardiac transplantation tolerance in wild-type B6 mice carrying a full mismatch BALB/c heart.     

CD4+CD25+ regulatory T cells in health and disease

1. Over the past 5 years, tremendous progress has been made in understanding the suppressive mechanisms of T regulatory (Treg) cells. The Treg cells, a subpopulation of T cells, have been shown to play an important role in maintaining peripheral tolerance and the prevention of autoimmunity. 2. Various populations of Treg cells have been described, including thymically derived CD4(+)CD25(+) Treg cells. These naturally occurring Treg cells are present in the periphery and are capable of suppressing proliferation and effector T cell responses both in vitro and in vivo. 3. In addition, a second subset of Treg cells, type 1 T regulatoary (Tr1) and Th3 cells, exert their suppressive capacity via cytokines such as interleukin-10 and transforming growth factor-beta and are contact independent. 4. The present review summarizes the characteristics and molecular basis of CD4(+)CD25(+) Treg cells, as well as their therapeutic potential in modulating inflammatory diseases, such as inflammatory bowel disease and rheumatoid arthritis.     

Development of TCR alpha beta CD8 alpha alpha intestinal intraepithelial lymphocytes is promoted by interleukin-15-producing epithelial cells constitutively stimulated by gram-negative bacteria via TLR4

The microbes present in the intestine have a strong influence on the development and maturation of lymphoid organs. The cross-talk mechanisms between intestinal intraepithelial lymphocytes (i-IEL) and noninvasive microbes are still poorly understood. The influence of microbes and lipopolysaccharides on the development of i-IEL, especially the TCR alpha beta(+) CD8 alpha alpha subset, was investigated using the different TLR4-mutant mouse strains C3H/HeJ, BALB/lps(d), and C57BL/10ScCr. Intestinal epithelial cells (i-EC) from TLR4-mutant strains did not express interleukin (IL)-15 mRNA, while IL-15 mRNA expression in i-EC from the corresponding wild-type, C3H/He, BALB/c, and C57BL/10ScSn mice was detected. The development of TCR alpha beta(+) CD8 alpha alpha cells in i-IEL significantly decreased in TLR4-mutant mice compared with the corresponding wild-type mice, while other T cell subsets in i-IEL showed similar percentages in the TLR4-mutant and wild-type mice. Adult thymectomized (ATx-) and lethally irradiated C3H/HeJ mice reconstituted with T cell-depleted bone marrow cells from C3H/He mice showed a significantly lower percentage of TCR alpha beta CD8 alpha alpha i-IEL than ATx-C3H/He mice after transfer of C3H/HeJ BM cells. The percentage of TCR alpha beta CD8 alpha alpha i-IEL and IL-15 mRNA expression in i-EC from BALB/lps(d) mice did not increase during Salmonella typhimurium infection but was significantly enhanced during Listeria monocytogenes infection. Our findings suggest that LPS induces IL-15 production by i-EC, resulting in the development of TCR alpha beta CD8 alpha alpha i-IEL.