Characterization of Lymphocyte Subpopulations and T Cell Activation In Endometriosis

PROBLEM: Numerous studies have characterized the lymphocyte subpopulations in normal eutopic endometrium and suggested a role for the cytokine secretory products of these lymphocytes in regulating endometrial cell proliferation and differentiation. Recent studies have shown that ectopic endometrium contains a greater concentration of scattered stromal lymphocytes than does eutopic endometrium. However, the lymphocyte subpopulations and their activation status have not been characterized in ectopic endometrium. METHODS: We performed immunohistochemical studies on serial sections of proliferative and secretory phase eutopic endometrium and ectopic endometrium obtained during the proliferative phase using monoclonal antibodies to CD4 (T helper-inducer cells), CD8 (T cytolytic-suppressor cells), CD22 (B-cells), CD56 (natural killer cells), and VLA-1 (T-cell activation marker). RESULTS: Ectopic endometrium contained significantly more scattered stromal CD4, CD8, and activated T cells than did proliferative and secretory eutopic endometrium. There were more activated T-cells in proliferative than in secretory eutopic endometrium. Ectopic endometrium contained significantly fewer NK cells than proliferative and secretory endometrium. CONCLUSIONS: These results demonstrate that (1) the increased lymphocyte population in ectopic endometrium is due to increased numbers of CD4 and CD8 cells, and (2) a greater number of activated T cells are present in ectopic endometrium as compared to eutopic endometrium. Increased concentration of stromal T cells and enhanced VLA-1 expression in ectopic endometrium suggest that cytokine products of the activated T-cells may be involved in regulating cellular processes of endometriosis tissue.     

慢性乙型肝炎患者外周血CD45RA+、CD45RO+T淋巴细胞亚群的特点及临床意义

目的：探讨慢性乙型肝炎患者外周血CD4^＋CD45RA^＋、CD4^＋CD45RO^＋、CD8^＋CD45RA^＋和CD8^＋CD45RO^＋T淋巴细胞亚群的特点及其与肝病病情的关系。方法：采集46例轻中度慢性乙型肝炎患者、58例重度慢性乙型肝炎患者和30例健康人的外周抗凝血，应用流式细胞技术三色荧光分析法对其外周血中CD4^＋CD45RA^＋、CD4^＋CD45RO^＋、CD8^＋CD45RA^＋和CD8^＋CD45RO＋T淋巴细胞亚群进行检测。结果：轻中、重度慢性乙型肝炎患者与正常人相比，其外周血中CD4^＋、CD8^＋T细胞均无明显改变；CD8^＋CD45RA^＋T细胞均明显降低，CD8^＋CD45RO^＋T细胞均明显增高，而CD4^＋CD45RA^＋、CD4^＋CD45RO^＋T细胞均无明显改变；重度慢性乙型肝炎患者与轻中度慢性乙型肝炎患者相比，CD8^＋CD45RA^＋T细胞明显降低（P〈0.05），CD8^＋CD45RO^＋T细胞明显升高（P〈0.05）。结论：乙型肝炎慢性化过程中，CD8^＋CD45RO^＋T细胞起重要作用且与慢性乙型肝炎患者病情的进展呈正相关；检测CD4^＋CD45RA^＋、CD4^＋CD45RO^＋、CD8^＋CD45RA^＋和CD8^＋CD45RO^＋T淋巴细胞亚群比检测CD4^＋和CD8^＋T细胞亚群更能正确、充分、全面地了解慢性乙型肝炎的发病机制和预后，从而有效地指导临床治疗。     

急性心肌梗塞患者外周血T淋巴细胞表达CD25及其对刺激剂反应能力的变化

目的：研究急性心肌梗塞患者外周血T淋巴细胞表达CD25及其对刺激剂反应能力的变化。方法：流式细胞仪技术。结果：急性心肌梗塞患者外周血淋巴细胞CD25 、CD4 和CD4 ／CD8 比值明显低于对照组,CD8 高于对照组;CD3 CD25 、CD4 CD25 和CD4 CD25 /CD8 CD25 显著降低。在培养前后,CD3 CD25 表达均显著降低;在PDB刺激情况下,CD3 CD25 、CD8 CD25 表达均显著降低。结论：急性心肌梗塞患者存在细胞免疫功能紊乱,并存在对刺激剂PDB的反应能力降低。     

Phenotypic and functional studies of leukocytes in human endometrium and endometriosis

The aetiology of endometriosis, a common and disabling disorder, is presently unknown, although immune dysfunction could allow ectopic endometrial fragments to survive outside the uterine cavity. These studies investigate the relationship between leukocyte populations, steroid hormone receptor expression, proliferative activity, bcl-2 expression and apoptosis in eutopic and ectopic endometrium from women with endometriosis or adenomyosis at different phases of the menstrual cycle. Significantly increased oestrogen receptor expression, bcl-2 expression and numbers of CD8+ leukocytes were found in ectopic compared with eutopic endometrium in endometriosis, and CD56+ endometrial granulated lymphocytes (eGLs) were significantly reduced in ectopic endometrium. Apoptotic cells were rarely found in control and subject endometria. In contrast with endometriosis, adenomyotic lesions showed identical steroid hormone receptor expression, proliferative activity, bcl-2 expression and leukocyte subpopulations to eutopic endometrium, indicating different aetiologies for these disorders. The unusual CD56+ CD16- eGLs present in large numbers in late secretory phase eutopic endometrium were highly purified (>98%) by immunomagnetic separation. Except for a negligible cytotoxic activity of eGLs from early proliferative samples, cytotoxic activity of eGLs from non-pregnant endometrium during the menstrual cycle was comparable with those in peripheral blood, predominantly CD56+ CD16+ natural killer cells. eGLs from non-pregnant endometrium and early pregnancy showed a variable proliferative response to 5 and 100 U/ml interleukin-2 over 48-h and 120-h time courses. eGLs are evidently functionally important in the eutopic endometrium. Their absence in endometriotic lesions together with increased CD+8 T-cell numbers and increased oestrogen receptor and bcl-2 expression may have significant effects on the development and progression of endometriosis.     

Lymphocyte immunophenotype reference ranges in healthy Indian adults: implications for management of HIV/AIDS in India

With the advent and application of standard and sensitive flow cytometry methods, it became essential to establish reference intervals in healthy individuals to demarcate between health and disease. A reference range of lymphocyte populations for normal individuals is important in the diagnosis and prognosis of immunodeficiency diseases like AIDS. We tried to accomplish this by studying the values for T lymphocyte subsets for 200 healthy North Indian adults between 18 and 55 years. We obtained the following reference ranges for various T lymphocyte subsets: CD4 count (304-1864 cells/microl with the median of 666 cells/microl), CD4% (17.5-50.6% with the median of 35%), CD8% (14-53% with the median of 32.3%), CD3% (43-89% with the median of 70.5%), and CD4/CD8 ratio (0.04-3.5 with the median of 1.04). Significant variations were observed for normal reference intervals for T lymphocyte subsets according to the race, ethnic origin, age group, and gender.     

Immunology: Bone marrow-derived cell populations in uterine and ectopic endometrium

Uterine endometrium contains numerous bone marrow-derived cells.The spectrum of cell types is different from that of any othertissue, and the differences in endometrium from women with endometriosismay reflect a different endometrial phenotype in these women.The cell types of bone marrow origin found in ectopic endometriummay indicate the degree of differentiation of the tissue. Itwas found that, in normal endometrium, the CD45⁺ cell populationcomprised T cells, macrophages, CD56⁺ large granular lymphocytes,some CD16⁺ cells and a few B cells. Changes in these cell populationsduring the menstrual cycle were similar in endometrium fromboth controls and patients with endometriosis, and resembledthat reported previously by others. In ectopic endometrium,the frequency of CD45⁺ cells remained within the same rangeas that of uterine endometrium but without any obvious patternof change during the menstrual cycle. CD56⁺ large granular lymphocytes,an immune cell type characteristic of uterine endometrium, werealso found in ectopic endometrium. Our results indicate thatectopic endometrium, as well as comprising both glandular andstromal cells, contains bone marrow-derived cell populationssimilar to those of uterine endometrium. This suggests thatthe same processes of cell migration and/or differentiationoccur in ectopic and uterine endometrium.     

Peritoneal natural killer cytotoxicity and CD25+CD3+ lymphocyte subpopulation are decreased in women with stage III-IV endometriosis

We examined the lymphocytes of peripheral blood (PB) and peritonealfluid (PF) of women with or without endometriosis to investigatethe alteration of cytotoxic activity of natural killer (NK)cells and activation of T cells in the peritoneal cavity ofwomen with endometriosis. A total 16 control women and 14 patientswith stage III–IV endometriosis were selected on the basisof laparoscopic examination in National Taiwan University Hospital.The lymphocyte subpopulations (B cell, NK cell and T cell),including T-cell activation markers (CD69, CD25, HLADR), inPB and PF were analysed by dual-colour flow cytometry. The NKcytotoxicity of PB and PF mononuclear cells was evaluated by⁵¹Cr release assay. There was significant decrease of NK cytotoxicityand CD25⁺CD3⁺lymphocyte subpopulation in PF of women with endometriosiscompared with those without endometriosis. However, there wasno difference in the proportion of NK cells in both PB and PFbetween women with and without endometriosis. Therefore, thedecreased NK cytotoxicity PF of women with endometriosis wasdue to the functional defect, but not quantitative defect, ofNK cells. The concomitant reduction of activated T cells inwomen with endometriosis might suggest its possible role inthe defect of NK cytotoxicity.     

T lymphocyte subset profile and serum alpha-1-antitrypsin in pathogenesis of chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) is an inflammatory disorder characterized by the presence of non-fully reversible airflow limitation. The study was undertaken to investigate the involvement of alpha-1-antitrypsin (alpha(1)AT) and T lymphocyte subsets in the pathogenesis of COPD. Blood samples of 50 subjects, including 25 healthy volunteers and 25 patients with COPD, were analysed. Serum trypsin inhibitory capacity (STIC) was determined by enzymatic assay. CD4(+) and CD8(+) T lymphocytes were enumerated in heparinized blood using a fluorescence activated cell sorter counter. The STIC in COPD patients was found to be decreased significantly than in controls (P < 0.01). In COPD patients with lower expression levels of alpha(1)AT, a highly significant decrease in the number of CD4(+) T lymphocytes (P < 0.0009) and CD4/CD8 ratio was observed compared with control subjects (P < 0.008). The mean +/- standard error of CD8(+) lymphocytes was found to be little different (only marginally decreased) in COPD patients compared to healthy controls; however, an alteration in the individual count of CD8(+) lymphocytes cells was observed in COPD patients. Using linear regression analysis, a negative correlation was observed between STIC and CD4(+) lymphocytes and CD8(+) lymphocytes (r = -0.40, P < 0.04; r = -0.42, P < 0.03, respectively) in COPD patients. An alteration in alpha(1)AT and T lymphocyte subsets in COPD patients suggested that interplay of these factors may be responsible for the progression of COPD.     

中国恒河猴(Macaca mulatta)外周血CD4+CD25+T淋巴细胞的研究

目的:研究中国恒河猴外周血中CD4 CD25 T淋巴细胞亚群及其分布频率。方法:利用流式细胞术对50只中国恒河猴外周血CD4 CD25 T淋巴细胞进行了分析。结果:发现所有被检测的恒河猴个体中均存在明显的CD4 CD25 T淋巴细胞亚群;CD4 CD25 T淋巴细胞大约占CD4 T淋巴细胞的9.1%(变化范围为2.6%～18.1%);其中CD4 CD25highT淋巴细胞约占2.5%(0.3%～5.5%)。对不同年龄和性别个体中CD4 CD25 T淋巴细胞频率的初步分析未发现统计学上有年龄或性别差异。结论:中国恒河猴可用于与CD4 CD25 T细胞相关的人类疾病的研究中。     

Longitudinal follow-up of lymphocyte subsets during the first year of life

A longitudinal study of lymphocyte subsets during infancy was evaluated by using the flow cytometric immunophenotyping method. Two hundred and thirteen blood samples were obtained from 92 healthy, full-term infants of the following ages: 1-7 days old (n = 43), 3 months old (n = 55), 6 months old (n = 57) and 11 months old (n = 58). The absolute numbers of CD3+ and CD3+/CD4+ T lymphocytes increased from birth to 3 months of age, and remained stable thereafter. The absolute number of CD3+/CD8+ T lymphocytes increased from birth to 11 months of age. The absolute number of CD19+ B lymphocytes and NK cells increased rapidly (3 months) after birth and continued to increase throughout the study period. However, the changes in the relative counts of lymphocyte subsets did not always correspond with the changes in their absolute numbers. These results demonstrate the age-related changes in lymphocyte subpopulations and provide reference ranges for lymphocyte subsets during infancy.     

Lymphocyte Subsets and Mitogen Stimulation of Blood Lymphocytes in Normal Pregnancy

PROBLEM: In normal pregnancy the maternal immune system should be directed towards tolerance or suppression in order not to reject the partly foreign feto-placental unit. The aim of this investigation was to find hallmarks of systemic immunosuppression during normal pregnancy. METHODS: Five healthy primigravidae were examined during pregnancy and postpartum with flow cytometric analysis to define T and B lymphocyte subsets in peripheral blood. In addition, we studied the proliferative response of lymphocytes to mitogens or interleu-kin-2 (IL-2) alone or in combination with immunomodulating drugs or interleukin-4 (IL-4). The results were compared to healthy, non-pregnant women. RESULTS: During pregnancy and early puerperium we noted an immune balance in favour of suppression, as measured by increased numbers of T “helper/suppressor” (CD4+ CD45RA+) and “suppressor”/effector T cells (CD8+S6F1-), and decreased numbers of T “helper/inducer” (CD4+CD29+), T “helper/memory” (CD4+CD45RO+), killer/effector T cells (CD8+S6F1+), and Natural Killer cells (CD56+), as well as decreased numbers of activated lymphocytes expressing IL-2 receptor (CD25+) and T cells expressing HLA-DR (HLA-DR+CD3+). During pregnancy, lymphocyte proliferation was impaired in autologous serum with concanavalin A (ConA), phytohemagglutinin (PHA), or IL-2. A difference in proliferative response to PHA or IL-2 between cultures with AB serum and autologous serum is suggestive of an immunosuppressor factor in serum during pregnancy. Indomethacin significantly increased lymphocyte proliferation in autologous serum with ConA, indicating PGE₂ mediated suppressor activity during pregnancy. Chlorambucil and cimetidine modulated the proliferative response to ConA, indicating an alkylating agent sensitive and a histamine dependent suppressor activity during pregnancy. CONCLUSIONS: During normal pregnancy, a state of systemic suppression of the maternal immune system seems to be present.     

交感神经对兔子宫内CD4＋T淋巴细胞分布和早期胚胎发育的影响

目的:手术切断支配兔子宫的交感神经后,观察其对子宫局部免疫状况和早期胚胎发育的影响.方法:使用H-E染色和免疫组织化学的方法观测子官内膜形态及CD4 T淋巴细胞数量及分布.结果:切断交感神经后,兔受孕率下降,胚胎着床数减少约61%,早期胚胎发育迟缓,甚至有死胚现象.组织学观察显示切断交感神经的妊娠兔子官内膜固有层不发达,明显未发育到适应胚泡着床的状态;子宫内CD4 T淋巴细胞发生了重新分布,肌层和非妊娠部位子官内膜的CD4 T淋巴细胞分布增多,而妊娠部位子宫内膜的CD4 T淋巴细胞数量减少.结论:交感神经通过调节子宫内膜组织学变化和子宫内CD4 T淋巴细胞的数量及分布,从而影响胚胎着床和早期发育.     

Changes in lymphocyte subpopulations and CD3+/DR+ expression in sepsis

Objective   To detect lymphocyte subpopulations and CD3+/DR + expression in sepsis.
Methods   In a prospective clinical study we evaluated subpopulations of lymphocytes and percentage of CD3⁺/HLA-DR⁺ lymphocytes using two-color flow cytometry in 40 patients with sepsis and compared them with 34 healthy adults.
Results   Septic patients, when compared with healthy controls, have significantly lower percentage and absolute numbers of total T lymphocytes and CD4 T lymphocytes ( P  < 0.01). Absolute numbers of CD8 T lymphocytes, NK cells, CD3+/DR + lymphocytes and CD4/CD8 ratio were also decreased ( P  < 0.01). The percentage of B lymphocytes was increased ( P  < 0.01).
Conclusion   Our results are in agreement with previous findings in patients with sepsis after major surgery or trauma. The decreases in the percentage and absolute numbers of circulating lymphocyte subsets in non-surgical sepsis could represent a general reaction to stress. Increased percentage of B lymphocytes is most probably related to the bacterial etiology of the disease.     

Elevated NK Cell Cytotoxicity,CD158a Expression in NK Cells and Activated T Lymphocytes in Peripheral Blood of Women with IVF Failures

Citation Chernyshov VP, Sudoma IO, Dons’koi BV, Kostyuchyk AA, Masliy YV. Elevated NK cell cytotoxicity, CD158a expression in NK cells and activated T lymphocytes in peripheral blood of women with IVF failures. Am J Reprod Immunol 2010; 64: 58–67 Problem The aim of this study was to evaluate the role of elevated natural killer cytotoxicity (NKc) in women with multiple implantation failures (IF) in vitro fertilization–embryo transfer (IVF–ET) cycles. Methods of study Seventy‐nine antiphospholipid antibodies‐negative women with IF including 33 women with elevated NKc were selected for investigation. K‐562 cell line was used to evaluate NKc. Lymphocyte subsets, intracellular cytokines [interferon (IFN)‐γ, interleukin (IL)‐4, tumour necrosis factor, IL‐10], expression of activating markers [CD69, human leukocyte antigen (HLA)‐DR], CD8, KIR (CD158a), CD95, and chemokine receptors (CXCR3, CCR4) were estimated by flow cytometry. Results In women with IF, levels of NKc were higher than in IVF successful women. IF was associated with higher expression of CD8, CD158a, and HLA‐DR in NK cells, activating markers in T lymphocytes, and lower levels of CCR4+ and IL‐4+ T lymphocyte subsets. Predictive value of single elevated NKc for IVF success was 0.85, but addition of two other abnormal parameters resulted in its decrease to <0.39. Conclusions Elevated NKc is negative factor, though not critical for implantation in IVF cycles. Immune mechanism of IVF failure includes not only elevated NKc but also some other factors, such as elevated expression of CD8 and CD158a on NK cells, T lymphocyte activation, and diminished T helper 2 parameters.     

多发性硬化症发作期外周血和脑脊液淋巴细胞亚群的分析

为分析多发性硬化症 (MS )患者发作期淋巴细胞亚群及给予甲基强的松龙 (MP )治疗后的变化 ,流式细胞仪测定 2 6例处于复发期MS患者外周血 (PB )和脑脊液 (CSF )及 8例MS患者予MP治疗后PB淋巴细胞CD3+ 、CD4 + 、CD8+ 、CD4 5RA+ 、CD4 + /CD4 5RA+ 、CD4 + /CD2 9+ 、CD19+ 、CD5 + /CD19+ 的百分率。结果发现MS患者PB中CD8+ 、CD4 5RA+ 和CD4 + /CD4 5RA+ 百分率降低 ,CD4 + /CD2 9+ 百分率和CD4 + /CD8+ 比值升高 ;CSF中CD3+ 、CD4 + 、CD4 + /CD2 9+ 百分率和CD4 + /CD8+ 比值高于PB ;淋巴细胞亚群与临床伤残程度和距此次发作的时间无关 ;MP治疗不影响PB淋巴细胞亚群变化。表明MS患者淋巴细胞通过血脑屏障有选择性 ,淋巴细胞亚群的变化在MS发病机制中起作用     

淋巴细胞共刺激信号和凋亡信号异常与RA免疫效应亢进的研究

目的：探究T淋巴细胞表面多种细胞信号分子所介导的细胞活化或凋亡信号在RA患者免疫功能紊乱中的作用。方法：采用流式细胞术检测RA患者外周血T细胞亚群及其表面共刺激分子cD154（cD40L）、CD30和凋亡受体CD95（Fas）的表达。结果：RA患者外周血T细胞亚群偏移，CD4^＋T细胞增加，CD8^＋T细胞减少；共刺激分子CD154在CD4^＋和CD8^＋T细胞上的表达均上调，但CD30分子的表达均降低，并以CD4^＋T细胞降低更为明显。同时，凋亡受体CD95分子在T细胞亚群上的表达均明显增加。结论：RA患者T淋巴细胞表面多种信号分子表达异常，共同导致了RA患者免疫功能紊乱。     

慢性乙肝病毒携带者外周血皮质醇水平与T细胞亚群状态的研究

目的 探讨慢性乙肝病毒(HBV)携带者血浆皮质醇水平与细胞免疫状态的变化.方法 选取符合诊断标准的慢性HBV携带者60例,正常对照10例,放射免疫法测定血浆皮质醇水平,流式细胞仪检测外周血T细胞亚群CD3+、CD4+、CD8+、CD4+/CD8+比例.结果 与正常对照组相比,慢性HBV携带者血浆皮质醇水平相对升高(P＜0.05),CD4+细胞比例无明显变化(P＞0.05),CD8+细胞比例明显增加(P＜0.05),CD4+/CD8+明显下降(P＜0.05).HBeAg阳性组与HBeAg阴性组比较,HBeAg阳性组血浆皮质醇升高较为明显(P＜0.05),CD4+细胞比例无明显变化(P＞0.05).CD8+细胞比例明显增加(P＜0.05),CD4+/CD8+明显下降(P＜0.05).结论 慢性乙肝病毒携带者存在着血浆皮质醇水平的增高及细胞免疫功能的失衡,慢性HBV感染过程中存在内分泌-免疫系统紊乱.     

TNF-alpha-induced apoptosis in neonatal lymphocytes: TNFRp55 expression and downstream pathways of apoptosis

Previously we have shown decreased Fas-mediated apoptosis in cord blood lymphocyte subsets. In this study, we compared tumor necrosis factor (TNF)-alpha-induced apoptosis in T lymphocytes and their subsets between cord blood and peripheral blood from healthy young controls. The expression of TNF receptor I (TNFR-I) was assessed by flow cytometry and quantitative RT-PCR. The expression of adapter molecules TNF receptor-associated death domain (TRADD), Fas-associated death domain (FADD) and TNF-associated factor-2 (TRAF-2) and caspase 3 was analyzed by Western blotting. The activity of caspase 3 and caspase 8 was measured by colorimetric assay. The susceptibility of CD4+ and CD8+ T cells to TNF-alpha-induced apoptosis was measured by terminal deoxytidyl transferase-mediated dUTP nick end labelling (TUNEL) assay. Both CD4+ and CD8+ T cell subsets from cord blood demonstrated decreased susceptibility to TNF-alpha-induced apoptosis that was associated with decreased activation of both caspase 8 and caspase 3 as compared to T cell subsets in peripheral blood. Furthermore, expression of TNFR-I, TRADD and caspase 3 was decreased in cord blood lymphocytes as compared to peripheral blood lymphocytes. The significance of these observations is discussed.     

中药狼疮方对狼疮样BXSB小鼠脾脏免疫系统和淋巴细胞亚群的影响

目的：研究中药狼疮方对狼疮样BXSB小鼠免疫系统和淋巴细胞亚群的影响，探讨狼疮方治疗SLE的免疫学机制。 方法： 采用BXSB小鼠模型，随机分为3组：狼疮方治疗组（每天0.5 mL狼疮方药液灌胃）、强的松治疗组（每天prednisone 0.173 mg/20 g BW溶于0.5 mL生理盐水灌胃），未治疗组（每天0.5 mL生理盐水灌胃），每组6只，疗程10周。另设与BXSB小鼠同基因的正常C57BL/6小鼠6只为正常对照组。分别采集上述各组小鼠外周血和脾组织进行检测。 结果： （1）未治疗组BXSB小鼠血清IgG和抗ds-DNA抗体水平及脾组织CD3、CD4、CD8、CD19、CD23阳性细胞百分比都显著高于正常对照组、狼疮方治疗组、强的松治疗组（P＜0.05或P＜0.01）。（2）经狼疮方或强的松治疗后，BXSB小鼠血清IgG、抗ds-DNA抗体水平及脾组织CD4、CD8、CD19、CD23阳性细胞百分比显著低于未治疗组（与未治疗组相比，P＜0.05或P＜0.01），且接近正常水平（与正常对照组比较，P＞0.05）。 结论： 狼疮样BXSB小鼠T、B细胞免疫功能上调。中药狼疮方可抑制狼疮样小鼠T、B淋巴细胞活化，减轻其高丙种球蛋白血症，减少体内自身抗体产生。