妊娠期间脾脏T淋巴细胞的改变与鼠胚着床

妊娠期间脾脏Ｔ淋巴细胞的改变与鼠胚着床温勇刘斌（北京医科大学组织学与胚胎学系）本实验应用流式细胞计测定方法，观察妊娠期间小鼠脾脏Ｔ淋巴细胞亚群的变化，研究母体适应胚胎着床的免疫调节，特别是脾脏在这一调节过程中的反应及意义。方法：分别于小鼠妊娠第２，６...     

小鼠胸腺基质细胞系(MTEC1)在裸鼠体内生物学特性研究

通过流式细胞仪（ＦＡＣＳ）、组织学染色和免疫组化的方法研究了脾内注射的小鼠胸腺上皮细胞系ＭＴＥＣ１和初代培养的小鼠胸腺基质细胞在裸鼠脾脏内存活状态及功能特性。结果发现ＭＴＥＣ１细胞不仅能够在脾脏内存活，而且在ＭＴＥＣ１细胞周围有大量不成熟的淋巴细胞。初代培养的小鼠胸腺基质细胞则不能在脾脏内存活。     

小鼠胸腺基质细胞系（MTEC1）在裸鼠体内生物学特…

通过流式细胞仪，组织学染色和免疫组化的方法研究了脾内注射的小鼠胸腺上皮细胞系ＭＥＥＣ１和初代培养的小鼠胸腺基质细胞在裸鼠脾脏内存活状态及功能特性。结果发现ＭＴＥＣ１细胞不仅能够在脾脏内存活，而且在ＭＴＥＣ１细胞周围大量不成熟的淋巴细胞。初代培养的小鼠胸腺基质细胞则不能在脾脏内存活。     

仙茅多糖对小鼠免疫功能调节作用实验研究

仙茅多糖体外单独能刺激小鼠脾淋巴细胞增殖，而单独对小鼠胸腺细胞无作用，但在ＣｏｎＡ存在条件下对胸腺细胞增殖有协同作用，体外对尼龙毛柱分离小鼠脾Ｔ细胞富含部分有明显刺激增殖作用：体外能对抗由氢化可的松（ＨＣ）抑制ＣｏｎＡ诱导脾Ｔ细胞增殖，体内对ＨＣ诱导免疫受抑小鼠胸腺及脾脏重量降低，胸腺细胞及脾Ｔ、Ｂ细胞增殖降低有明显对抗作用。     

卡介克蟹对巨噬细胞功能的调变

卡介克蟹是卡介苗的提取物，应用ＭＴ法研究了卡介克蟹对巨噬细胞功能的调变作用。结果显示：卡介克蟹能直接活化巨噬细胞；增强小鼠腹腔巨噬细胞的细胞毒作用；促进ＩＬ－１的分泌；并能增强脾脏介导的脾脏Ｔ淋巴细胞ＣｏｎＡ增殖反应。     

同种特异T细胞疫苗诱导移植物存活期延长的研究：Ⅲ.抗T细胞抗…

本文观察了ＴＣＶ免疫小鼠血清对同种抗原和非特异抗原刺激的淋巴细胞反应性的影响，结果显示，ＴＣＶ免疫小鼠血清能显著抑制ＣｏｎＡ诱导的淋巴细胞增殖和同种ＭＬＲ，表明该血清中存在有抑制Ｔ细胞功能的“抑制因子”实验还发现ＴＣＶ免疫小鼠血清中存在针对多种不同抗原决策簇抗体，包括有ＴＣＶ特异的，活化Ｔ细胞的和正常淋巴细胞的，ＴＣＶ呼吸实验证明ＴＣＶ免疫小鼠血清中能特异地与ＴＣＶ结合的抗体不能与其它活化了Ｔ细胞     

免疫小鼠GALT部位T淋巴细胞回忆反应观测

以鼠伤塞杆菌减毒Ｇ３０经口免疫小鼠两次，间隔３或５个月后再刺激，应用间接免疫荧光法检测了小鼠派伊尔氏淋巴结，肠系膜淋巴结及脾脏中Ｌ３Ｔ４、ＬＹＴ２Ｔ细胞亚群变化并以ＭＴＴ法测定了ＣｏｎＡ诱导的淋巴细胞增反应。     

中药“强盾”冲剂用于继发性细胞免疫缺陷小鼠的实验研究

本文研究了具有益气养阴补育中药“强盾”冲剂（ＱＤ）对糖皮质激素（ＡＣＳ）所致继发性细胞免疫缺陷小鼠的免疫恢复作用。结果表明，ＱＤ对免疫损伤后小鼠的白细胞计数、淋巴细胞绝对值、Ｔ淋巴细胞亚群百分比及脾脏、胸腺的重量均出现明显恢复现象（Ｐ＜０．００１）。还发现ＱＤ与ＡＣＳ同时应用，有拮抗ＡＣＳ免疫损伤的作用（Ｐ＜０．００１）。ＱＤ对正常状态下免疫尚未发现明显的促进作用。     

雷公藤抑制致敏小鼠CD4^＋T细胞核因子GATA3,NFAT的活性

目的 探讨致敏小鼠ＣＤ４＾＋Ｔ淋巴细胞核转录因子ＧＡＴＡ３、ＮＦＡＴ活性的变化及雷公藤内酯醇（ＴＰ）的作用。方法 采用卵蛋白（ＯＶＡ）致敏的方法建立模型；运用ｐａｎｎｉｎｇ法分离ＣＤ４＾＋Ｔ淋巴细胞；通过凝胶电泳迁移率实验（ＥＭＳＡ）对ＣＤ４＾＋Ｔ淋巴细胞核因子ＧＡＴＡ３、ＮＦＡＴ的ＤＮＡ结合活性及ＴＰ的作用进行检测，同时就ＴＰ的作用与地塞米松（ＤＭ）相比较。结果 正常小鼠ＣＤ４＾＋Ｔ淋巴细胞核因     

ICAM-1在着床期小鼠PBLC及EC/DC中表达的研究

目的：研究细胞间粘附因子－１（ＩＣＡＭ－１）在着床期外周血淋巴细胞（ＰＢＬＣ）、子宫内膜及蜕膜细胞（ＥＣ／ＤＣ）中的不同表达特点及动态变化规律。方法：以着床期小鼠为动物模型,细胞分析采用单抗免疫荧光标主多参数流式细胞术分析技术。结果：发现ＩＣＡＭ－１在ＰＢＬＣ、ＥＣ／ＤＣ中的均存在明显的动态变化;其中ＩＣＡＭ－１的表达在ＰＢＬＣ中于妊娠第２天（Ｄ２）达最低小平,而在ＥＣ／ＤＣ中于妊娠第４天（Ｄ４）     

T and B lymphocyte subsets in patients with unexplained recurrent spontaneous abortion: IVIG versus placebo treatment

PROBLEM: To investigate circulating lymphocyte subsets in women with recurrent spontaneous abortion (RSA) in relation to pregnancy outcome and to treatment with intravenous immunoglobulin (IVIG). METHOD OF STUDY: Forty-one women with a history of unexplained RSA were examined during first trimester of pregnancy before IVIG or placebo treatment and after pregnancy. The results were compared with five healthy, non-pregnant women and five women in the first trimester of normal pregnancy. Circulating lymphocyte subsets with focus on T-cell subpopulations were determined by flow cytometry. RESULTS: The proportions of human leukocyte antigen (HLA)-DR positive T cells (CD3+ HLA-DR+), T-killer/effector cells (CD8+ S6F1+) and B cells (CD19+) were increased, whereas the proportion of T-suppressor/inducer cells (CD4+ CD45RA+) was decreased during first trimester pregnancy of RSA women compared with pregnant normal controls. T and B lymphocyte subsets did not correlate with pregnancy outcome on either IVIG or placebo group. CONCLUSIONS: In RSA patients, the immune system seems to be activated in contrast to the suppression noted in normal pregnancy.     

Changes of Lymphocyte Subsets in Normal Pregnant and Postpartum Women: Postpartum Increase of NK/K (Leu 7) Cells

ABSTRACT: Changes in lymphocyte subsets in whole blood of normal pregnant and postpartum women were examined by flow cytometry with an automated leukocyte differential system. From the first trimester and throughout pregnancy, the absolute counts of T(CD3) and B(CD20) and T-cell subsets (CD4, CD8) decreased with a decrease in the absolute lymphocyte count, although the proportions of these cells remained unchanged except for a decrease in the percentage of T helper-inducer (CD4) cells in the first trimester. On the contrary, the percentage of NK/K (Leu 7) cells, but not of NK/K (CD16) cells, increased in the first trimester and then both gradually decreased in the second and third trimesters. In the postpartum period, the percentages and absolute counts of T(CD3) and NK/K (Leu 7) cells, but not of other cells, increased transiently. These changes of lymphocyte subsets may indicate suppression of immunological activity during pregnancy and its “increase” in the postpartum period.     

Immunoregulatory factors in multiple sclerosis patients during and after pregnancy: relevance of natural killer cells

Multiple sclerosis (MS) ameliorates typically during pregnancy but after the delivery the relapse rate often increases. Our study was conducted to understand the immunoregulatory mechanisms accompanying this phenomenon. MS patients were followed-up prospectively during pregnancy and 6 months postpartum, with immunological characterization of the peripheral blood. Groups of age- and parity-matched healthy pregnant women, and age- and sex-matched non-pregnant women and non-pregnant MS patients were studied as controls. In our patient cohort, the annualized relapse rate was 1.0 +/- 1.0 relapses/woman/year (mean +/- standard deviation) during the year before pregnancy, but dropped to 0.2 +/- 0.9 during the third trimester (P = 0.02). After the delivery the relapse rate increased again to 1.4 +/- 1.9 (1-3 months postpartum versus third trimester P = 0.003). While percentages of peripheral blood CD3, CD4, CD8 and CD19 immune cell subsets were unchanged over the observation period, reduced disease activity during the last trimester was associated with a significant increase in the percentage of circulating CD56(bright) natural killer (NK) cells. Simultaneously, the proportion of circulating CD56(dim) NK cells was clearly reduced. No alteration was noted in CD4+ CD25(high) forkhead box P3+ regulatory T cells. Production of interferon-gamma by peripheral blood lymphocytes was down-regulated significantly during pregnancy in comparison to the postpartum period, resulting in an increased T helper type 2 (Th2) : Th1 ratio during pregnancy. In conclusion, pregnant state in MS patients is characterized by an increase in the percentage of CD56(bright) NK cells and by enhanced Th2 type cytokine secretion. Our findings suggest a potential role for CD56(bright) regulatory NK cells in the control of autoimmune inflammation during pregnancy in MS.     

慢性肾炎患者外周血T细胞亚群和共刺激分子的表达及其意义

为研究慢性肾炎患者外周血T细胞亚群和共刺激分子的表达特点及其在慢性肾炎免疫病理机制中的作用 ,本文采用免疫荧光标记和流式细胞仪分析 ,对 35例慢性肾炎患者外周血T淋巴细胞亚群和共刺激分子CD2 8、 4 1BB等的表达进行研究。结果表明 :(1)慢性肾炎患者T细胞亚群明显失衡 ,表现为CD4减少 ,CD8增加 ,CD4/CD8比值显著降低 ;(2 )共刺激分子CD2 8表达显著低于正常对照组 (CD2 8表达百分率分别为 45 95± 5 6 7和 6 6 42± 4 5 8,P <0 0 0 1) ,且CD4+ CD2 8+ T细胞和CD8+ CD2 8+ T细胞均显著减少。治疗后缓解期患者T细胞亚群失衡明显纠正 ,CD2 8+ T细胞 ,尤其是CD4+ CD2 8+ T细胞显著增多 ,而且CD4+ CD2 8+ T细胞数与患者的 2 4h尿蛋白定量呈负相关 (r= 0 47,P <0 0 1) ;(3)慢性肾炎患者共刺激分子 4 1BB在T细胞中的表达显著高于正常对照组 (表达百分率分别为 30 5 7± 8 12和 0 74± 0 2 8,P <0 0 0 1) ,治疗后的 4 1BB表达水平显著降低 ,而且 4 1BB异常高表达与CD8+ T细胞数呈正相关 (r=0 6 3,P <0 0 5 )。从而表明慢性肾炎外周血T细胞亚群失衡和T细胞活化所必需的共刺激分子CD2 8、 4 1BB异常表达 ,可能在慢性肾炎发生和病变进展中起着重要作用。     

妊娠期调节性T细胞介导保护性效应需要同种抗原的刺激

越来越多的证据表明,人和小鼠妊娠期CD4+CD25+调节性T细胞(Treg)参与介导母胎免疫耐受。然而,目前对于妊娠期间调控Treg变化的因素还不清楚,对同基因交配(BALB/c×BALB/c)和异基因交配(BALB/c×C57)小鼠以及妊娠妇女CD4+CD25+Treg进行了分析,以探讨雌、孕激素和胎儿同种异基因抗原对Treg的影响。发现异基因孕鼠外周淋巴器官CD4+CD25+T细胞于早孕、中孕期明显高于非孕对照,而在同基因孕鼠只有少数个例升高;这些CD4+CD25+T细胞均表达Foxp3蛋白,在体外具有无反应性和免疫抑制能力;而且异基因孕鼠淋巴细胞对父方同种抗原的应答强度明显弱于同基因孕鼠。对去势的雌鼠给予雌二醇或孕酮,使其在血清中浓度接近中孕水平,未发现外周血CD4+CD25+Treg的明显变化。妊娠妇女CD4+CD25highTreg在早孕、中孕期显著升高,临近分娩期降至非孕水平,而此时CD4+CD25lowT细胞反而增加。以上结果表明,异基因抗原的存在对于妊娠期CD4+CD25+Treg介导对胎儿的免疫保护效应具有重要作用,而CD4+CD25+Treg的消失可能与分娩的发动有一定关系。     

The Status of Tumor Immunology and Cancer Immunotherapy

To explore if the increased percentages of Regulatory T (Treg) cells, as well as, overexpression of Cytotoxic T-Lymphocyte Antigen-4 (CTLA-4) are involved in laryngeal-squamous cell carcinoma (SCC), 45 patients with laryngeal-SCC and 27 healthy controls were enrolled. Flow cytometry was performed to investigate, in the peripheral blood, the prevalence of CD4+CD25+FoxP3+ Treg cells, as well as, surface and intracellular expression of CTLA-4 by the main lymphocyte subsets (CD4+, CD8+ and CD19+). The results indicated intracellular (In)CTLA4 with considerable higher expression in the CD8+ lymphocytes among patients with laryngeal-SCC compared with the control group (8.2 ± 8.7 versus 2.3 ± 3.5, P = 0.001). The mean percentage of InCTLA4+CD4+ and InCTLA4+CD19+ lymphocytes was also significantly higher in patients (8.7 ± 7.8 versus 4.4 ± 4.2, P = 0.018 and 0.6 ± 0.8 versus 0.2 ± 0.2, P = 0.024, respectively). With respect to surface (Sur)CTLA4, the difference between patients and controls was, however, significant only in the case of CD8+ lymphocytes (0.7 ± 0.6 versus 0.3 ± 0.3, P = 0.003, respectively). The percentage of Treg cells was observed to be significantly higher in patients (7.5 ± 6.3 and 3.2 ± 1.9, P < 0.0001). Furthermore, association analysis revealed the association of Treg cell increase with the higher tumor-size and lymphnode stage (P < 0.005). These data collectively suggest that patients with laryngeal-SCC may benefit from immunotherapy targeting CTLA4 and Treg cells.     

Increased Frequencies of the CD29 and CD57 Markers and Decreased Frequency of CD45RA Within CD4+ and CD8+ Subsets after Allogeneic Bone Marrow Transplantation in Man

Two monoclonal antibodies, anti-CD45RA and anti-CD29, reciprocally divide the CD4⁺ and CD8⁺ lymphocytes into CD4⁺ CD45RA⁺, CD4⁺ CD29⁺, CD8⁺ CD45RA⁺ and CD8⁺ CD29⁺ subsets. The CD4⁺ CD45RA⁺, CD4⁺ CD29⁺ and CD8⁺ CD45RA⁺ possess suppressor-inducer, helper-inducer and suppressor-effector functions respectively. Since the role of these subsets has not been established after allogeneic bone marrow transplantation we studied lymphocyte subpopulations in 12 patients 45- 227 days after the procedure. The fraction of CD4⁺ lymphocytes was significantly (P= 0.0005) decreased to 20±9% versus 43±3% in controls. Within the CD4⁺ compartment, we found an increase in the fraction of CD4⁺ cells that co-expressed CD29 (CD29⁺/CD4⁺) to 92±10% versus 48±15% (P=0.008) in controls and a concommittant decrease m CD45RA⁺/CD4⁺ to 16±12% versus 56±25% (P=0.008) Patients were also noted to have an increase in the percentage of CD8⁺ lymphocytes to 41±5% compared to 23±4% in controls (P=0.0004), Examination of the CD8⁺ subsets revealed a significant increase in the CD29⁺/CD8⁺ fraction to 97±3% versus 64±2% in controls (P= 0.008) and a decrease in the CD45RA⁺/CD8⁺ fraction to 36±11% versus 70±21% (P= 0.008). The number of cells co-expressing CD57 were also determined within the CD4⁺ and CD8⁺ subsets. In patients CD57⁺/CD4⁺ were increased to 29±7% versus 1±1% in controls (P=0.04), and CD57⁺/CD8⁺ to 49±12% versus 23±9% (P=0.02). Since CD29⁺ and CD57⁺ cells have a poor capability for IL-2 production and proliferation this shift in subset distribution may account for some of the defects in cellular immunity seen within the first year after allogeneic bone marrow transplantation.     

Effect of Pregnancy on Thymic T Cell Development

PROBLEM: The thymus gland decreases in size during pregnancy. The significance of this alteration is not known. METHOD: In this report, we examined thymic function by evaluating the development of T lymphocytes in the thymus of pregnant Balb/c mice at 15 and 20 days gestation using multi-color flow cytometry. Comparative analysis was made with non-pregnant mice, postpartum lactating mice, and postpartum non-lactating mice. RESULTS: Progressive reduction of thymic size and cellularity during pregnancy was observed. All of the CD4 and CD8 defined subsets were reduced, with a disproportionate loss of CD4+, CD8+ double positive cells. Examination of the CD4-, CD8- double negative compartment revealed a predominance of TCR α,β+ double negative cells, and a striking loss of precursor cells. The CD3-, CD4-, CD8- triple negative thymic subset was composed almost entirely of the earliest population (CD44+, CD25-), with the remaining maturational stages (CD44+, CD25+; CD44-, CD25+; and CD44-, CD25-) depleted. At 2 weeks postpartum, the subset ratios normalized, and the total cell count showed recovery. CONCLUSION: T cell development is blocked at the precursor level during the mouse pregnancy. These effects are transient, and gradual recovery is observed in the postpartum period.     

Alleviation of chronic GVHD in mice by oral immuneregulation toward recipient pretransplant splenocytes does not jeopardize the graft versus leukemia effect

Chronic graft versus host disease (cGVHD) is the result of an immune-mediated attack by transplanted donor lymphocytes, entailing inflammatory damage to host target organs. Clinically, the post-bone marrow transplantation (BMT) graft versus leukemia (GVL) effect may be associated with GVHD. Immune hyporesponsiveness induced by oral antigen administration has recently been shown to prevent the development of cGVHD in a murine model. To evaluate whether amelioration of cGVHD in mice by induction of oral immune regulation in donors toward recipient pretransplant lymphocyte antigens is associated with attenuation of the GVL effect donor B10.D2 mice were fed with Balb/c splenocytes, B10.D2 splenocytes, bovine serum albumin (BSA), or regular chow, every other day for 10 days. Subsequently, transplantation of 2 x 10(7) splenocytes from donor B10.D2 mice to recipient Balb/c mice was undertaken, followed by inoculation of 3 x 10(3) BCL-1 leukemia on the day of BMT. Control groups were fed identically without leukemia inoculation. Mice were followed for survival and leukemia progression. Induction of tolerance was assessed by a mixed lymphocyte reaction (MLR). Cutaneous GVHD was assessed macroscopically. To elucidate the mechanism of any observed effect, serum interferon (IFN), interleukin (IL-2), IL-12, IL-4, and IL-10 were determined by enzyme-linked immunosorbent assay and flow cytometry analysis for CD4+, CD8+, and NK1.1+ lymphocyte subpopulations was performed. There was no significant difference in leukemia progression manifested by survival or white blood cell counts of orally immune-regulated mice compared with control animals. Cutaneous cGVHD was significantly ameliorated in Balb/c mice transplanted from tolerized B10.D2 mice. This effect was associated with a significant reduction in the mixed lymphocyte response of effector splenocytes from tolerized B10.D2 mice against Balb/c target splenocytes; significantly decreased serum IFN-gamma and IL-2; increased serum IL-12 levels; increased peripheral NK1.1+ cells; and CD4+/CD8+ lymphocyte ratio. Oral tolerization of BMT donors toward recipient antigens ameliorates cGVHD without hampering the GVL effect.     

Low circulating CD4(+) CD25(+) Foxp3(+) T regulatory cell levels predict miscarriage risk in newly pregnant women with a history of failure

Citation Winger EE, Reed JL. Low circulating CD4⁺ CD25⁺ Foxp3⁺ T regulatory cell levels predict miscarriage risk in newly pregnant women with a history of failure. Am J Reprod Immunol 2011; 66: 320–328 Problem The purpose of this study was to determine whether quantification of peripheral blood Treg cell levels could be used as an indicator of miscarriage risk in newly pregnant women with a history of immunologic reproductive failure. Method of Study Fifty‐four pregnant women with a history of immunologic infertility and/or pregnancy loss were retrospectively evaluated (mean age: 36.7 ± 4.9 years, 2.8 ± 2.5 previous miscarriages; 1.5 ± 1.9 previous IVF failures). Twenty‐three of these women experienced another first trimester miscarriage, and 31 of these women continued their current pregnancies past 12 weeks (‘pregnancy success’). The following immunologic parameters were assessed in the first trimester: NK cell 50:1 cytotoxicity, CD56⁺ 16⁺ CD3^? (NK), CD56⁺ CD3⁺ (NKT), TNFα/IL‐10, IFNγ/IL‐10, CD4⁺ CD25^?Foxp3⁺, total CD4⁺ Foxp3⁺ (CD4⁺ CD25⁺ Foxp3 plus CD25^? Foxp3⁺), and CD4⁺ CD25⁺ Foxp3⁺ levels. Results Patients with successful ongoing pregnancies experienced a mean (CD4⁺ CD25⁺ Foxp3⁺) ‘Treg’ level of 0.72 ± 0.52%, while those that miscarried in the first trimester experienced a mean Treg level of 0.37 ± 0.29% (P = 0.005). Markers not significantly different between the loss and success groups were NK 50:1 cytotoxicity (P = 0.63), CD56⁺ 16⁺ 3⁺ NK cells (P = 0.63), CD56⁺ 3⁺ NKT (P = 0.30), TNFα⁺IL‐10⁺(P = 0.13), IFNg⁺IL‐10⁺ (P = 0.63), and CD4⁺ 25^? Foxp3⁺ cells (P = 0.10), although total CD4⁺ Foxp3⁺ levels remained significant (P = 0.02) and CD4⁺ 25⁺ Foxp3⁺ showed the most significant difference (P = 0.005). Mean day of blood draw was 49.2 ± 36.1 days pregnant (median 39.0 days). In addition, patients with a low Treg level (<0.7%) in the first trimester experienced a significantly lower ongoing pregnancy rate than those with a higher Treg level (>0.7%) in the first trimester [44% (15/34) versus 80% (16/20); P = 0.01]. Of the 18 successful pregnancies with sequential Treg results, 85% (11/13) showed a T‐regulatory‐cell‐level increase (mean Treg change 0.33 ± 0.32), while only 40% (2/5) of the failed pregnancies showed a Treg increase (mean Treg change ?0.08 ± 0.28; P = 0.02). Conclusions From these data, we propose that CD4⁺ CD25⁺ Foxp3⁺ T regulatory cells may serve as a superior pregnancy marker for assessing miscarriage risk in newly pregnant women. Larger follow‐up studies are needed for confirmation.