SLE患者CD45RA^＋和CD45RO^＋T细胞亚群的变化

利用流式细胞术及免疫双荧光染色法，检测３５例系统性红斑狼疮（ＳＬＥ）患者外周血ＣＤ４５ＲＡ和ＣＤ４５ＲＯＴ细胞亚群，结果表明：ＳＬＥ患者ＣＤ４细胞降低，ＣＤ８细胞增高，ＣＤ４／ＣＤ８降低，ＣＤ４ＣＤ４５ＲＡ细胞在病情活动期降低，在稳定期回升，ＣＤ４ＣＤ４５ＲＯ细胞在活动期有增高倾向，在稳定期下降，ＣＤ８ＣＤ４５ＲＡ及ＣＤ８ＣＤ４５ＲＯ细胞均在活动期增高，又均在稳定期降至正常水平，提示ＣＤ４５ＲＡ和     

自身免疫病患者T淋巴细胞表型分析

本文报告利用流式细胞仪检测了１０例系统性红斑狼疮（ＳＬＥ）患者、１８例干燥综合症（ＳＳ）患者、３７例风湿性关节炎（ＲＡ）患者和１６例人外周血Ｔ淋巴细胞表型，发现ＳＬＥ患者ＣＤ３＾＋Ｔ细胞明显降低，ＳＳ和ＲＡ患者正常；ＳＬＥ患者ＣＤ４＾＋／ＣＤ８＾＋Ｔ细胞比值倒置，ＳＳ和ＲＡ正常，但ＳＳ和ＲＡ ＣＤ８＾＋Ｔ细胞明显减少。文中对这三种疾病发病机理进行了初步讨论。     

GM-CSF诱导外周血单核细胞的分化及其对混合淋巴细胞反应的影响

作者对 Ｇ Ｍ Ｃ Ｓ Ｆ 作用下的外周血单核细胞（ Ｐ Ｍ Ｃ） 的形态特征及免疫表型进行了测定, 并观察了其对混合淋巴细胞反应（ Ｍ Ｌ Ｒ） 的影响。结果显示, Ｇ Ｍ Ｃ Ｓ Ｆ 可使外周血单核细胞分化为 Ｃ Ｄ１４ ＋ 、 Ｃ Ｄ１ａ 或低表达、 Ｃ Ｄ８０ 、 Ｃ Ｄ８６ 低表达、 Ｈ Ｌ Ａ Ｄ Ｒ＋ 、 Ｃ Ｄ１１ａ ＋ 、 Ｃ Ｄ４５ Ｒ Ａ＋ , 和非特异性酯酶呈强阳性反应的细胞, 符合巨噬细胞的形态及表型特征。在 Ｍ Ｌ Ｒ 体系中加入上述细胞, 发现其 Ｍ Ｌ Ｒ 的结果因巨噬细胞含量的不同而异, 巨噬细胞与反应细胞比例为１∶２ 时抑制 Ｍ Ｌ Ｒ（ Ｐ＜ ００５ ） , １∶４或１∶８ 时刺激 Ｍ Ｌ Ｒ（ Ｐ＜ ００５ ） ; Ｐ Ｈ Ａ 和 Ｉ Ｌ ２ 能够逆转大剂量巨噬细胞对 Ｍ Ｌ Ｒ 的抑制作用, 抗 Ｃ Ｄ３ 单抗可增强其对 Ｍ Ｌ Ｒ 的抑制作用, 消炎痛不影响巨噬细胞对 Ｍ Ｌ Ｒ 的作用。     

CD4^＋CD45^＋T细胞对SLE病人B细胞产生自身抗体的调节作用

选择体外抗ＤＮＡ，抗ＣＤＬ抗体双阳性的ＳＬＥ病人Ｂ细胞做为靶细胞，分别与不同的ＳＬＥ病人或正常人ＣＤ４＾＋ＣＤ４５＾＋Ｔ细胞亚群共同培养，结果显示，ＳＬＥ病人ＣＤ４＾＋ＣＤ４５ＲＯ＾＋Ｔ细胞对自身抗体的产生有明显的辅助作用，而ＣＤ４＾＋ＣＤ４５ＲＡ＾＋Ｔ细胞的作用不明显。     

痢疾杆菌口服免疫小鼠肠道相关淋巴组织中CD45R^＋和CD45RB^＋…

为了解肠道相关淋巴细胞（ＧＡＬＴ）对痢疾杆菌口服免疫的应答状况，分离了Ｓｈｉｇｅｌｌａｆｌｅｘｅｒｉ四次免疫后Ｂａｌｂ／ｃ小鼠脾（ＳＰ），肠系膜淋巴结（ＭＬＮ），Ｐｅｙｅｒ’ｓ结（ＰＰ），固有膜（ＬＰ）和上皮内（ＩＥ）淋巴细胞，对末次免疫后１～３ｄ内免疫组与对照组ＧＡＬＴ中的ＣＤ４５Ｒ＾＋和ＣＤ４５ＲＢ＾＋细胞群作了ＦＣＳ分析，结果免疫后起动７ｄＩＥ和ＬＰＣＤ４５Ｒ＾＋细胞增加而ＭＬＮ和ＰＰＣＤ４     

自身免疫性疾病患者T细胞表面抗原CD2,CD4和CD8mRNA的表达

用斑点杂交技术检测了１８例系统红斑狼疮（ＳＬＥ），２２例干燥综合征（ＳＳ）、２１例类风湿性关节炎（ＲＡ）患者及１６例正常人外周血淋巴细胞ＣＤ２，ＣＤ４和ＣＤ８ｍＲＮＡ的表达水平，结果表明，这３种病人ＣＤ２ｍＲＮＡ水平均较正常人有明显升高，ＳＬＥ和ＲＡ患者ＣＤ４／ＣＤ８ｍＲＮＡ比值也明显升高，而ＳＳ患者ＣＤ４／ＣＤ８ｍＲＮＡ比值在正常范围，在Ｔ细胞发育过程中，ＣＤ２先天ＣＤ４和ＣＤ８的表达，结果提示     

普通变异型免疫缺陷病CD45RA^＋和CD45RO^＋T细胞亚群异常

用不同的单抗可将Ｔ淋巴细胞区分为不同的功能亚群：“处女”Ｔ细胞（ｎａｉｖｅ，ＣＤ４５ＲＡ￣＋）及“记忆”Ｔ细胞（ｍｅｍｏｒｙ，ＣＤ４５ＲＯ￣＋）。以直接双标记免疫荧光经流式细胞仪观察了普通变异型免疫缺陷病ＣＤ４￣＋及ＣＤ８￣＋细胞中ＣＤ４５ＲＡ￣＋　和ＣＤ４５ＲＯ￣＋细胞亚群的分布及变化，发现ＣＤ４／ＣＤ８比值倒置的患者ＣＤ４￣＋ＣＤ４５ＲＡ￣＋Ｔ细胞亚群显著减少，而ＣＤ８￣＋ＣＤ４５ＲＯ￣＋和ＣＤ８￣＋ＣＤ４５ＲＡ￣＋亚群明显增高。     

异种移植中两条识别途径的细胞及分子基础研究初探

为探讨猪（供者）人（受者）异种移植时人Ｔ细胞的识别途径，以人ＰＢＭＣ为受者反应细胞，猪ＰＢＭＣ为供者刺激细胞建立异种ＭＬＲ模型，应用去除ＡＰＣｓ、３Ｈ－ＴｄＲ掺入、单克隆抗体封闭及ＦＡＣＳ分析方法，研究了供者（猪）和受者（人）ＡＰＣｓ及受者粘附分子在ＭＬＲ中作用，并动态观察了ＣＤ４＋和ＣＤ８＋细胞增殖变化。结果发现去除供者及受者ＡＰＣｓ均可降低异种ＭＬＲ（Ｐ＜０．０５），而以前者更为明显（Ｐ＜０．０１），若两者ＡＰＣｓ均去除，ＭＬＲ仅表现为弱阳性；并观察到ＣＤ４＋细胞自ＭＬＲ第１天开始逐渐升高，ＣＤ８＋细胞第３天开始升高。利用ＣＤ４、ＣＤ８、ＣＤ１１ａ、ＣＤ１８、ＣＤ５４及ＣＤ５８单抗封闭人淋巴细胞表面分子后，可不同程度抑制ＭＬＲ（Ｐ＜０．０１），而ＣＤ１１ｂ和ＣＤ４４无此作用（Ｐ＞０．０５）。本结果证实，Ｔ细胞异种识别中存在直接识别和间接识别途径，并首先活化ＣＤ４＋Ｔ细胞，且有多种粘附分子参与识别作用。     

IL-4在诱导CD4,45RA~ 和CD4,45RO~ T细胞向IL-4产生细胞分化过程中的效应

应用ＥＬＩＳＰＯＴ（Ｅｎｚｙｍｅ－ｌｉｎｋｅｄｉｍｍｕｎｏｓｐｏｔａｓｓａｙ）单细胞检测技术，研究了淋巴因子在诱导ＣＤ４，４５ＲＯ＋和ＣＤ４，４５ＲＡ＋Ｔ细胞向ＩＬ－４产生细胞分化过程中的影响。在存在ＰＭＡ和ｉｏｎｏｍｙｃｉｎ的情况下，ＩＬ－２和ＩＬ－４均可以诱导ＣＤ４，４５ＲＯ＋Ｔ细胞分化为ＩＬ－４产生细胞，但是只有ＩＬ－４可以诱导ＣＤ４，４５ＲＡ＋Ｔ细胞向ＩＬ－４产生细胞分化，ＩＦＮγ既不能诱导ＣＤ４，４５ＲＡ＋，亦不能对ＣＤ４，４５ＲＯ＋Ｔ细胞产生诱导作用。当内源性ＩＬ－４被中和后，ＩＬ－２对ＣＤ４，４５ＲＯ＋Ｔ细胞的诱导作用消失。本文结果初步表明ＩＬ－４在决定Ｔｈ前体细胞是向Ｔｈ１还是向Ｔｈ２方向发展起着关键的作用。     

慢性阻塞性肺病患者细胞粘附分子表达研究

对慢性阻塞性肺病（ＣＯＰＤ）患者外周血单个核细胞（ＰＢＭＣｓ）表面的ＣＤ１１ａ／ＣＤ１８，ＣＤ１１ｂ／ＣＤ１８（ＡＰＡＡＰ法）及ＣＤ４４（流式细胞分析法）粘附分子表达及血浆中可溶怀Ｅ．Ｐ．－选择素水平进行检测。结果：ＣＰＯＤ急性加重期患者ＰＢＭＣｓ表面ＣＤ１１ａ、ＣＤ１１ｂ及ＣＤ４４分子表达明显增高，血浆中可溶性Ｅ．Ｐ－选择素水平亦显著增高，与正常人及缓解期患者相比均有显著性差异。综合治疗可使血浆     

Quantitative Analysis of Lymphocyte CD1 la using Standardized Flow Cytometry

CDlla is the alpha-subunit of the leucocyte adhesion and costimulation molecule LFA-1. We have refined the measurement of lymphocyte CDlla density with a FACScan using commercially available fluorescent beads for standardization and fluorescein-conjugated antibody to CDlla of known fluorescein:protein ratio. The fluorescence intensity of CDlla on peripheral blood CD4⁺ and CD8⁺ lymphocytes was measured in 60 healthy subjects. We demonstrated linear correlation between age and mean CDlla density (r =0.47 for CD4⁺ cells, r = 0.71 for CD8⁺ cells). We established that there is a consistent logical cut off point at 4.3 × 10³ bound antibody molecules between low-expressing and high-expressing subsets of CD8⁺ cells and we then investigated whether the variation in lymphocyte CDlla expression in healthy subjects was sufficiently small for the application of this method to the detection of abnormal groups or individuals. Analysis of the CDlla high subsets has high statistical power (>99% in 60 subjects to detect a 25% difference) and good precision (<4% differences). The advantages of the method for comparative studies of cell surface accessory molecules are discussed. We have also evaluated a frozen cell line for quality control, and demonstrated up-regulation of CD1la density on CD4+ and CD8+ cells measured in three patients with infectious mononucleosis.     

CD45 isoform expression during T cell development in the thymus.

Various isoforms of leukocyte common antigen, or CD45, are expressed differentially on T cells at different stages of development and activation. We report studies on CD45 isoform expression on various subsets of human T cells using two- and three-color flow cytometry and cell depletion. Bone marrow cells that were depleted of CD3+ and HLA-DR+ cells were CD45RA-RO-. The earliest CD3-CD4-CD8-CD19- thymocytes were CD45RO- with 20%-30% CD45RA+ cells. The most prominent population of CD4+CD8+ double-positive thymocytes were CD45RA-RO+. Even the CD4+CD8+ blasts were greater than 90% CD45RO+. About 80% of single-positive thymocytes (CD4+CD8- or CD4-CD8+) were also CD45RO+. Only 4.3% of CD4+ and 18% of CD8+ single-positive thymocytes were CD45RA+. In contrast, cord blood T cells which represent the stage that immediately follows single-positive thymocytes, contained 90% CD45RA+ cells. Thus, in terms of CD45 isoform expression, single-positive thymocytes are more like double-positive cells than cord blood T cells. These results suggest the following sequence of CD45 isoform switching during T cell development: CD45RA-RO- or RA+RO- (double-negative thymocytes)----RA-RO+ (double-positive and most single-positive thymocytes)----RA+RO- (cord blood T cells), the last switch from CD45RO to CD45RA occurring as a final step of maturation in the thymus.     

Phenotypic changes associated with activation of CD45RA+ and CD45RO+ T cells.

Resting CD45RO+, mature/memory, T cells are phenotypically distinct from intermediate CD45RO+/CD45RA+ and CD45RA+, immature/virgin, T cells, and are characterized by high levels of expression of a number of adhesion molecules, such as CD2, CD18, CD58 and CD29. The kinetics of up-regulation of molecules, like CD25 and CD54 associated with activation, were similar in both subsets and suggested that their high level expression was associated with later events rather than initial recognition and signal transduction. CD45RA+ T cells, unlike CD45RO+ T cells, were unable to proliferate in response to mitogenic combinations of CD2 monoclonal antibodies (mAb), although in combination with submitogenic doses of PMA both up-regulation of cell-surface molecules and proliferation occurred. In addition, recruitment of CD45RA+ T cells by CD2 mAb-activated CD45RO+ T cells can occur.     

Up-regulated expression of Fas antigen (CD95) by peripheral naive and memory T cell subsets in patients with systemic lupus erythematosus (SLE): a possible mechanism for lymphopenia.

Fas antigen (CD95) is a membrane-associated molecule that mediates apoptotic cell death and may play a role in the induction and maintenance of T cell tolerance. To elucidate the involvement of Fas antigen in human autoimmune diseases, we analysed Fas antigen expression by peripheral T cells from patients with SLE and rheumatoid arthritis (RA), using three-colour flow cytometry. Both CD4+ and CD8+ T cells from SLE patients expressed Fas antigen in a higher density than did these cells from healthy donors and from RA patients. Enhancement of Fas antigen density was noted in Fas+CD45RO+ memory T cells from SLE patients. More remarkably, a significant expression of Fas antigen was observed in CD45RO- naive T cells from SLE patients. CD4+CD45RO- T cells from SLE patients co-expressed Fas antigen and early to intermediate activation antigens such as CD25 and CD71, and late activation antigen HLA-DR in only FashiCD4+ naive T cells. Such up-regulation of Fas antigen expression in SLE patients seems to be clinically meaningful, because mean fluorescence intensity (MFI) of Fas antigen on CD4+ T cell subsets inversely correlates with the absolute size of CD4+ T cell subsets in peripheral blood of SLE patients. These results suggest that T cells with increased Fas antigen expression may be highly susceptible to apoptotic cell death, in vivo. A putative mechanism for lymphopenia in SLE patients is discussed.     

Dual expression of CD45RA and CD45RO isoforms on myelin basic protein-specific CD4+ T-cell lines in multiple sclerosis

Myelin basic protein (MBP)-specific T-cell lines from patients with multiple sclerosis (MS) and healthy controls were analyzed for the expression of CD45 isoforms and adhesion molecules. In the multiple sclerosis group, 22 of 24 MBP-specific T-cell lines were CD4+. Two distinct patterns were observed with regard to CD45 isoform expression. Pattern I showed dual expression of CD45 isoforms (CD4+CD45RA+CD45RO+CD29+) and Pattern II included cells with a single CD45 isoform (CD4+CD45RA–CD45RO+CD29+). All 10 cell lines from healthy controls were CD4+ and displayed Pattern II (CD4+CD45RA–CD45RO+CD29+). The dual expression of CD45 isoform in T-cell lines from MS was stable, did not represent a transition stage from CD45RA to CD45RO, and was cell-cycle independent. All cell lines from MS and controls expressed increased levels of LFA-1 (CD11a), LFA-2 (CD2), LFA-3 (CD58), ICAM-1 (CD54), and VLA-4 (CDw49d). These data show the presence of unique MBP-specific T cells (CD4+CD45RA+CD45RO+CD29+) that might play a role in the pathogenesis of MS.     

Blood levels of CD11b+ memory T lymphocytes are selectively upregulated in patients with active rheumatoid arthritis

The adhesion molecules CD11b (a beta2-integrin component) and CD54 (ICAM-1) on blood leukocytes were studied by flow cytometry in patients with rheumatoid arthritis (RA). The fractions of CD4+ cells co-expressing CD11b were elevated in 16 patients with active RA compared with those in 16 RA patients who improved during therapy and 8 healthy controls: 0.8+/-0.12% (mean+/-SEM) versus 0.3+/-0.06% (p<0.002) and 0.3+/-0.06% (p<0.005), respectively. Increased levels of CD11b+CD45R0+ cells were observed in patients with active RA compared to those with improved RA and controls: 12.6+/-3.9% versus 4.8+/-2.7% (p<0.002) and 6.1+/-1.2% (p<0.003), respectively. Disease activity, determined by C-reactive protein, correlated with the numbers of CD11b+CD45R0+ cells: r=0.62 (p<0.001). Seven patients were followed during induction of remission with methotrexate and glucocorticoids. The numbers of CD11b+CD4+ and CD11b+CD45R0+ cells fell significantly after clinical improvement. The levels of CD11b+CD14+ cells (monocytes) did not differ between the groups. The number of CD11b+CD15+ cells (neutrophils) was elevated in patients with RA irrespective of disease activity. The levels of CD54+ cells were not different between the RA and control groups. We conclude that the increased numbers of CD11b+ memory T cells may arise from exposure to stimuli outside the synovial compartment.     

Human CD8+ intraepithelial T lymphocytes are mainly CD45RA-RB+ and show increased co-expression of CD45R0 in celiac disease

Expression of various CD45 isoforms (RA, RB and R0) on CD3+, CD4+ and CD8+ intraepithelial and lamina propria T cells was examined in situ by a three-color immunofluorescence technique in jejunal biopsy specimens from 32 patients with celiac disease and 18 controls. The median percentage of CD3+ intraepithelial lymphocytes (IEL) that expressed CD45R0 increased from 52% in controls to 69% in untreated celiac disease (p less than 0.01). Furthermore, the percentages of CD4+ and CD8+ IEL strongly positive for CD45R0 rose respectively from 94% and 24% in controls to 100% and 55% in untreated celiac disease. Conversely, CD45R0 was strongly expressed on most CD3+ lamina propria lymphocytes (LPL) both in control (81%) and diseased (77%-81%) mucosa. A variable fraction of the intraepithelial and lamina propria CD3+ T cells expressed mainly CD45RB (controls, 46% and 20%, respectively; celiac disease, 29% and 15%). Only 2% IEL and 4% LPL were positive for CD45RA. Expression of different CD45R isoforms thus identified three distinct CD8+ T cell subsets in human intestinal mucosa. In addition, our results suggested that antigen-primed CD8+CD45R0+ memory cells accumulate in the jejunal epithelium of patients with untreated celiac disease.     

CD45RA antibodies split the CD3bright T cell subset.

Thymocyte subsets have been well characterized on the basis of CD4 and CD8 antigen expression. Recently, the use of anti-CD3 antibodies has allowed more precise phenotyping of these subsets. The most immature T cell precursors are largely CD3-CD4-CD8-, while the most mature are CD3brightCD4+CD8- or CD3brightCD4-CD8+. Moreover, the expression of CD45RA on thymocytes appears to define a progenitor population and may define a continuous lineage of cells. Using a panel of CD45RA antibodies, we have further characterized the CD45RA+ thymocyte population in the murine system. The size of this subset is greatly enhanced in cortisone-treated mice and in sublethally irradiated mice. Moreover, the CD45RA+ population is present early in foetal life and is maintained thereafter. Using three-colour immunofluorescence, we show that (i) while most CD45RA+ cells are present amongst the CD4-CD8- thymocyte subset in the normal thymus, after cortisone treatment or irradiation, all four thymocyte subsets co-express significant amounts of CD45RA. This suggests that not only progenitor cells but also the mature population which can survive such manipulation are CD45RA+; and (ii) a large proportion of CD45RA+ cells are CD3bright and this subset is represented in the thymus at all stages of maturation tested. These data suggest that a proportion of TCR-gamma delta + CD3+ cells in the fetus as well as of TCR-alpha beta+ CD3+ cells in the adult co-express CD45RA.     

CD45RO and CD45RA positive cell populations in idiopathic membranous and IgA glomerulopathy.

AIMS: To immunophenotype and quantitate glomerular and interstitial inflammatory cells in cases of idiopathic membranous and IgA glomerulopathy; to correlate cell numbers with aspects of clinical data and renal function. METHODS: Routine indirect immunoperoxidase staining was performed on frozen section renal biopsy specimens for T and B lymphocyte related antigens, macrophages and MHC class II antigens. Double immunohistochemical staining was performed to identify CD45RO+ and CD45RA+ cells. RESULTS: In IgA glomerulopathy correlations were found relating interstitial cell numbers to creatinine concentration at biopsy (CD45RO+ and CD45RA+ cells) and follow up creatinine concentration (CD3+, CD4+, CD8+, CD45RO+, and CD45RA+ cells). Also in IgA glomerulopathy mean arterial pressure at biopsy correlated with interstitial cell numbers and most recent follow up creatinine concentration. There were no correlations between glomerular inflammatory cells and renal function in either disease. Double staining showed that although most glomerular CD45RO+ and CD45RA+ cells were macrophages, positive cells in the interstitium were lymphocytes. The interstitial CD45RO+:RA+ ratio in normal renal biopsy specimens was approximately 5:1; for IgA glomerulopathy it was 1.5 and was 1.0 in idiopathic membranous glomerulopathy. CONCLUSIONS: This study demonstrates that interstitial, and not glomerular, inflammatory cell numbers correlate with renal function in primary glomerular disease and that double staining is necessary to interpret positive immunostaining for antigens located on more than one type of inflammatory cell. Detailed investigation of the interstitial CD45RO+ and CD45RA+ cells may give an insight into the pathogenesis of glomerular disease.