Stimulation of Human Peripheral Lymphocytes via CD3 and Soluble Antigen Abrogates Specific Antibody Production by Reducing Memory B Cell Numbers

Human B cells are polyclonally activated in vitro by T cells stimulated with immobilized anti-CD3 monoclonal antibodies. We have analysed the effect of CD3 ligation on the production of antigen-specific antibodies, using peripheral blood lymphocytes from tetanus toxoid vaccinated blood donors. High levels of antigen-specific antibodies were obtained after stimulation with anti-CD3 antibodies for 7 days. Addition of a soluble recall antigen did not affect the total amount of Ig produced, but dramatically decreased the antigen-specific response. The addition of IL-2, IL-4, anti-CD40 or anti-CD28 antibodies or the removal of antigen did not restore the B cell response. Analysis using limiting dilution of B cells showed that the frequency of antigen-specific memory B cells decreased significantly in cultures stimulated with antigen. The antigen-specific B cell response could be completely restored only if the soluble antigen was cross-linked on the surface of the B cells. These results suggested that peripheral memory B cells were eliminated or anergized in the presence of soluble antigen.     

重症肌无力患者CD4、CD8细胞表面Fas分子的变化

为探讨Fas分子、CD4、CD8分子在重症肌无力发生中的作用 ,从胸腺及外周血分离单个核细胞 ,用荧光标记的单克隆抗体 (Fas FITC、CD4 PE、CD8 Cy )和流式细胞仪检测细胞表面Fas、CD4、CD8表达情况。结果发现 ,(1)MG患者外周血单个核细胞 (PBMC )CD4 + CD8 细胞明显低于对照组 (P <0 0 2 ) ;(2 )MG患者Fas分子在PBMC的表达率明显低于对照组 (P<0 0 2 ) ,Fas+ CD4 + 细胞也低于对照组 (P <0 0 5 ) ;(3)在Fas+ 的胸腺细胞中 ,MG患者的双阳性细胞显著低于对照组 (P <0 0 1) ,而CD4 CD8+ 细胞则高于对照组 (P <0 0 5 )。说明重症肌无力患者淋巴细胞存在Fas表达异常 ,表现为外周CD4 + 细胞活化后的凋亡障碍和胸腺CD4 + CD8+ 细胞凋亡障碍 ,可能与重症肌无力患者自身反应性T细胞的形成及疾病的发生有关     

The Effect of Capping by Anti-immunoglobulin Antibody on the Expression of Cell Surface Immunoglobulin and on Lymphocyte Activation

The ability of mouse spleen cells to bind anti-mouse immunoglobulin antibody labelled with ¹²⁵I (anti-mouse Ig Ab ¹²⁵I) in vitro was measured. The validity of this technique for quantifying the amount of surface immunoglobulin on mouse spleen cells was established by showing that the specific uptake of anti-mouse Ig Ab ¹²⁵I was linearly related to the number of spleen cells present. The technique was used to assess the exposed immunoglobulin determinants on mouse spleen cells after the redistribution of their surface associated immunoglobulins to a polar cap (capping) had been induced by anti-mouse Ig Ab. It was found that after capping the amount of surface immunoglobulin recovered almost to control levels, but showed no further increase. Treatment of spleen cells with chymotrypsin and papain but not trypsin markedly reduced the quantity of surface immunoglobulin. The rate of immunoglobulin resynthesis after chymotrypsinisation was not affected by previously capping the surface immunoglobulins. Concentrations of anti-mouse Ig Ab which induced capping of the surface immunoglobulin of spleen cells did not stimulate them to lake up (³H) thymidine although under the same culture conditions spleen cells were activated by phytohaemagglutinin and lipopolysaccharide. It is considered that capping is not in itself a sufficient stimulus to bring about in spleen cells either an increase in the density of the surface immunoglobulin or the initiation of DNA synthesis.     

Soluble CD163, a Specific Macrophage Activation Marker,is Decreased by Anti‐TNF‐α Antibody Treatment in Active Inflammatory Bowel Disease

Activated macrophages shed the haemoglobin–haptoglobin scavenger receptor CD163 into the circulation as soluble(s)‐CD163. We measured sCD163 as an in vivo macrophage activation marker in patients with Crohn's disease (CD) or ulcerative colitis (UC) receiving antitumour necrosis factor (TNF)‐α antibody or prednisolone treatment. We also investigated the CD163 expression on circulating monocytes. 58 patients with CD, 40 patients with UC and 90 healthy controls (HC) were included. All patients had active disease at inclusion and were followed for 6 weeks of anti‐TNF‐α antibody or prednisolone treatment. We measured plasma sCD163 levels at baseline, 1 day, 1 week and 6 weeks after initiating treatment. CD163 expression on circulating CD14⁺ monocytes was measured in 21 patients with CD receiving anti‐TNF‐α antibody treatment. Baseline sCD163 levels were elevated in patients with CD [1.99 (1.80–2.18) mg/l] and in patients with UC [2.07 (1.82–2.32) mg/l] compared with HC [1.51 (1.38–1.63) mg/l] (P < 0.001). Anti‐TNF‐α antibody treatment induced a rapid decrease in sCD163 levels in patients with CD and in patients with UC 1 day after treatment initiation (P < 0.05). One week of prednisolone treatment did not induce a reduction in sCD163 levels. Anti‐TNF‐α treatment normalized sCD163 levels in patients with UC, whereas patients with CD exhibited sustained increased sCD163 levels. In patients with CD, CD163 expression on CD14⁺ monocytes was increased compared with HC. This study highlights that active CD and UC are associated with increased macrophage activation, as indicated by elevated sCD163 levels and monocytic CD163 expression. Anti‐TNF‐α antibody treatment induced a rapid decrease in sCD163 levels, suggesting a specific effect on macrophage activation in inflammatory bowel diseases.     

大鼠骨髓基质细胞表面抗原CD71、CD44和CD45的检测

目的研究表面抗原CD71、CD44和CD45在体外培养的大鼠骨髓基质细胞的表达,了解骨髓基质细胞的生物学特性。方法采用全骨髓法分离培养大鼠骨髓基质细胞,待原代培养的细胞达70%以上汇合时,用含胰酶和EDTA的消化液消化细胞,进行传代培养。传至第3代(P3)的骨髓基质细胞采用流式细胞术检测表面抗原CD71、CD44和CD45的表达,专用配套软件计算细胞表面抗原阳性%率。结果骨髓基质细胞呈纺锤形、成纤维细胞样生长,漩涡状分布。传至第3代的细胞形态趋于一致。流式细胞仪检测结果显示,CD71、CD44和CD45阳性细胞百分比分别为99.07±7.6%、52.81±7.3%和10.85±5.0%。结论P3的骨髓基质细胞具有较强的增殖特性。     

Cell Survival and Cytokine Release after Inflammasome Activation Is Regulated by the Toll-IL-1R Protein SARM

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Expression of Costimulatory Molecules CD80 and/or CD86 by a Kaposi's Sarcoma Tumor Cell Line Induces Differential T-Cell Activation and Proliferation

During physiological stimulation of resting T-cells, at least two activation signals by antigen presenting cells are required. Besides the first antigen-specific signal, the second costimulatory signal involves CD80 and CD86 expressed by the antigen presenting cell. These costimulatory molecules have been suggested to be of clinical relevance in many different autoimmune and malignant disease processes. We previously observed that tumor cells in Kaposi's sarcoma (a common AIDS-related cutaneous neoplasm) completely lack both CD80 and CD86, and these tumor cells fail to stimulate T-cell proliferation. In this study, using a Kaposi's sarcoma tumor cell line designated SLK, various stable transfected cell lines were produced. Tumor cells that were either singly positive for either CD80 or CD86, as well as a double-positive cell line, were examined for their ability to induce T-cell activation, T-cell proliferation, and cytokine production profiles. Compared to the parental double-negative tumor cell line, the CD80-positive cells, but not the CD86-positive tumor cells, induced significant T-cell activation and proliferation. Tumor cells expressing both CD80 and CD86 also induced T-cell activation. After stimulation by the transfected tumor cells, T-cells produced a Th-1 type cytokine production profile with increased IL-2 and IFN-gamma levels. These results demonstrate that Kaposi's sarcoma tumor cells lacking co-stimulatory molecules cannot induce T-cell activation; however, if they express CD80, they can induce peripheral blood T-cell proliferation, and there is a differential response as expression of CD86 did not have the same immunostimulatory effect.     

靶向CD20和HLA-DR分子Crossmab的设计与构建表达

目的设计、构建与表达靶向CD20和HLA-DR分子的Crossmab双特异性抗体。方法设计靶向CD20和HLA-DR分子的Crossmab双特异性抗体分子,命名为CD20-HLA-DR CrossmabCH1-CL,利用基因工程技术构建Crossmab四条链的真核表达载体,然后利用FreeStyle 293-F Cells哺乳动物细胞表达体系表达CD20-HLA-DR CrossmabCH1-CL。结果获得了CD20-HLA-DR CrossmabCH1-CL抗体分子蛋白。结论成功设计、构建、表达并初步鉴定了靶向CD20和HLA-DR分子的双特异性抗体CD20-HLA-DR CrossmabCH1-CL,为研制新型抗肿瘤的双特异性抗体药物打下了基础。     

The Release of Soluble Factors Contributing to Endothelial Activation and Damage after Hematopoietic Stem Cell Transplantation Is Not Limited to the Allogeneic Setting and Involves Several Pathogenic Mechanisms

This study evaluated the relative impact of the intensity of the conditioning regimen and the alloreactivity in the endothelial dysfunction occurring after allogeneic hematopoietic stem cell transplantation (allo-HSCT). It involved a comparative analysis of the effect of incubating human umbilical vein endothelial cells (ECs) with serum samples from patients receiving autologous HSCT (auto-HSCT) or unrelated donor allo-HSCT. In both groups, blood samples were collected through a central line before conditioning (Pre), before transplantation (day 0), and at days 7, 14, and 21 after transplantation. Changes in the expression of EC receptors and adhesion proteins, adhesion of leukocytes and platelets under flow, and signaling pathways were analyzed. Endothelial activation and damage were observed in both groups, but with differing patterns. All markers of endothelial dysfunction demonstrated a progressive increase from day Pre to day 14 in the auto-HSCT group and exhibited 2 peaks of maximal expression (at days 0 and 21) in the allo-HSCT group. Both treatments induced a proinflammatory state (ie, expression of adhesion receptors, leukocyte adhesion, and p38 MAPK activation) and cell proliferation (ie, morphology and activation of ErK42/44). Prothrombotic changes (ie, von Willebrand factor expression and platelet adhesion) predominated after allo-HSCT, and a proapoptotic tendency (ie, activation of SAPK/JNK) was seen only in this group. These findings indicate that endothelial activation and damage after HSCT also occur in the autologous setting and affect macrovascular ECs. After the initial damage induced by the conditioning regimen, other factors, such as granulocyte colony-stimulating factor (G-CSF) toxicity, engraftment, and alloreactivity, may contribute to the endothelial damage seen during HSCT. Further studies are needed to explore the association between this endothelial damage and the vascular complications associated with HSCT.     

SLE患者细胞粘附分子CD11a、CD18、CD54表达初探

利用流式细胞术及免疫荧光双染色法，检测３５例系统性红斑狼疮（ＳＬＥ）患者外周血细胞粘附分子表型（ＣＤ１１ａ、ＣＤ１８、ＣＤ５４）及淋巴细胞表型（ＣＤ３、ＣＤ４、ＣＤ８、ＣＤ４５、ＲＡ、ＣＤ４５ＲＯ）。结果表明，ＳＬＥ活动组ＣＤ８＋细胞增高，ＣＤ４＋ＣＤ４５ＲＡ＋细胞减少；ＣＤ４＋细胞表面ＣＤ１１ａ、ＣＤ１８表达降低，后二者在ＣＤ８＋细胞上表达增高；ＣＤ５４在ＣＤ２０＋细胞上增高。进一步发现，ＣＤ８＋细胞的ＣＤ１８增高与ＣＤ４＋ＣＤ４５ＲＡ＋细胞减少呈负相关（Ｐ＜００５），而与ＣＤ２０＋细胞表面ＣＤ５４增高呈正相关（Ｐ＜００１）。本文提示，细胞粘附分子可能在ＳＬＥ发病机理中占有重要意义。     

Critical stoichiometric ratio of CD4+ CD25+ FoxP3+ regulatory T cells and CD4+ CD25− responder T cells influence immunosuppression in patients with B‐cell acute lymphoblastic leukaemia

Regulatory T (Treg) cells act to suppress activation of the immune system and thereby maintain immunological homeostasis and tolerance to self-antigens. The frequency and suppressing activity of Treg cells in general are high in different malignancies. We wanted to identify the role and regulation of CD4⁺ CD25⁺ FoxP3⁺ Treg cells in B-cell acute lymphoblastic leukaemia (B-ALL). We have included patients at diagnosis (n = 54), patients in clinical remission (n = 32) and normal healthy individuals (n = 35). These diagnosed patients demonstrated a lower number of CD4⁺ CD25⁺ cells co-expressing a higher level of FoxP3, interleukin-10, transforming growth factor-β and CD152/CTLA-4 than the normal population. Treg cells from patients showed a higher suppressive capability on CD4⁺ CD25⁻ responder T (Tresp) cells than normal. The frequency and immunosuppressive potential of CD4⁺ CD25⁺ FoxP3⁺ Treg cells became high with the progression of malignancy in B-ALL. Relative distribution of Tresp and Treg cells was only ˜5 : 1 in B-ALL but ˜35 : 1 in normal healthy individuals, further confirming the elevated immunosuppression in patients. A co-culture study at these definite ex vivo ratios, indicated that Treg cells from B-ALL patients exhibited higher immunosuppression than Treg cells from normal healthy individuals. After chemotherapy using the MCP841 protocol, the frequency of CD4⁺ CD25⁺ cells was gradually enhanced with the reduction of FoxP3, interleukin-10 positivity corresponded with disease presentation, indicating reduced immunosuppression. Taken together, our study indicated that the CD4⁺ CD25⁺ FoxP3⁺ Treg cells played an important role in immunosuppression, resulting in a positive disease-correlation in these patients. To the best of our knowledge, this is the first detailed report on the frequency, regulation and functionality of Treg cells in B-ALL.     

TNF-α Regulates GM-CSF-, IL-3- or M-CSF-Induced Fc ε RII/CD23 Gene Expression and Soluble Fc ε RII Release by Human Monocytes

The authors examined the regulatory effects of tumour necrosis factor-α (TNF-α) on granulocyte macrophage colony stimulating factor (GM-CSF)-, interleukin-3 (IL-3)- or macrophage colony stimulating factor (M-CSF)-induced gene expression of the low affinity receptor for IgE (Fc ε RII) on human monocytes and GM-CSF-, IL-3- or M-CSF-induced soluble Fc ε RII (sFc ε RII) release from monocytes. The expression of GM-CSF-, IL-3- or M-CSF-induced Fc ε RII on the surface of monocytes was reduced by TNF-α. The present analysis was designed to examine whether or not TNF-α could suppress GM-CSF-, IL-3- or M-CSF-induced Fc ε RII messenger RNA (mRNA) expression and enhance the release of sFc ε RII induced by these cytokines. The addition of TNF-α to monocyte cultures with GM-CSF, IL-3 or M-CSF significantly reduced Fc ε RII expression on the surface of monocytes and significantly increased sFc ε RII release from monocytes. These results suggest that TNF-α-dependent reduction of GM-CSF-, IL-3- or M-CSF-induced Fc ε RII expression on the surface of monocytes resulted, at least in part, from the suppression of Fc ε RII mRNA and the enhancement of sFc ε RII release.     

Concentrations of Cytokines, Soluble Interleukin-2 Receptor, and Soluble CD30 in Sera of Patients with Hepatitis B Virus Infection during Acute and Convalescent Phases

The immunoregulatory roles of interleukin-2 (IL-2), IL-4, IL-10, gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), the soluble form of the IL-2 receptor (sIL-2R), and the soluble form of CD30 (sCD30) were evaluated in patients with hepatitis B virus (HBV) infection. Two groups of subjects were studied: 15 healthy individuals without hepatitis antecedents and 15 patients with HBV infection. Blood samples were taken during the acute and convalescent phases. The analysis of the samples was done by the enzyme-linked immunosorbent assay technique. IFN-γ and TNF-α levels decreased in the convalescent phase. IL-10, IL-2, and sIL-2R levels increased in the acute and convalescent phases, while sCD30 levels increased during the acute phase. The IL-4 concentrations decreased in both phases. During the acute phase, IFN-γ and TNF-α induced increases in IL-2, sIL-2R, IL-10, and sCD30 levels in serum, which allowed the development of immunity characterized by the nonreactivity of the HBV surface antigen, the onset of antibodies to the HBV surface antigen (anti-HBs), and normal alanine aminotransferase levels during the convalescent phase. Increased IL-2 levels during the acute phase would stimulate the activities of NK cells and CD8⁺ lymphocytes, which are responsible for viral clearing. The raised sIL-2R levels reveal activation of T lymphocytes and control of the IL-2-dependent immune response. The sCD30 increment during the acute phase reflects the greater activation of the Th2 cellular phenotype. Its decrease in the convalescent phase points out the decrease in the level of HBV replication. The increase in IL-10 levels could result in a decrease in IL-4 levels and modulate IFN-γ and TNF-α levels during both phases of disease, allowing the maintenance of anti-HBs concentrations.     

慢性乙型病毒性肝炎患者外周血T淋巴细胞CD27和CD45RA表达的研究

目的分析慢性乙型病毒性肝炎患者外周血T淋巴细胞CD27和CD45RA的表达。方法采集分离健康人和慢性乙型病毒性肝炎患者外周血单个核细胞(PBMC),利用多种荧光标记抗体标记细胞表面分子,再用流式细胞仪检测CD8+T淋巴细胞表面CD27和CD45RA表达情况。结果31例慢性乙型病毒性肝炎患者CD8+CD45RA+CD27+T细胞占CD8+T细胞(29.03±13.18)%,低于28例健康对照组的(60.85±14.36)%,P<0.01。而CD8+CD45RA-CD27+T细胞占CD8+T细胞(30.31±24.11)%,显著高于健康对照组的(10.32±5.24)%,P<0.05。慢性乙型病毒性肝炎患者CD4+CD45RA+CD27+T细胞21.12±9.64%低于健康对照组的(60.89±17.93)%,P<0.01,而CD4+CD45RA-CD27+T细胞(54.28±18.75)%显著高于健康对照组的(27.16±9.24)%,P<0.01。结论健康人外周血CD8+和CD4+T淋巴细胞均以CD45RA+CD27+初始细胞表型为主,而慢性乙型病毒性肝炎患者外周血初始细胞减少,CD45RA-CD27+表型的T淋巴细胞明显增加。     

Activation of Rat Alveolar Macrophage-Derived Latent Transforming Growth Factor β-1 by Plasmin Requires Interaction with Thrombospondin-1 and its Cell Surface Receptor, CD36

Transforming growth factor-beta-1 (TGF-beta1) is secreted by cells in a latent form (L-TGF-beta1) noncovalently bound to a latency-associated peptide. Activated alveolar macrophages obtained from rat lungs after bleomycin-induced pulmonary injury released increased amounts of active TGF-beta1 as well as plasmin, a protease, and thrombospondin-1 (TSP-1), a trimeric glycoprotein. Previously we had demonstrated that plasmin was critical to the activation of L-TGF- beta1. In the present study we demonstrated that TSP-1 is also important for the activation of L-TGF- beta1 because the activation can be inhibited by anti-TSP-1 monoclonal antibody. Proteins obtained from alveolar macrophage cell lysates immunoprecipitated with antibodies specific for TSP-1 were identified on immunoblots as LAP and TGF-beta1, indicating that TSP-1/L-TGF-beta1 complexes are present on alveolar macrophages. However, in the presence of plasmin both latency-associated peptide and TGF-beta1 were decreased in the same cell lysates, indicating that L-TGF-beta1 associated with TSP-1 is released by plasmin. Using immunofluorescence and antibodies to TGF-beta1 and CD36, a receptor for TSP-1, there was colocalization of TGF-beta1 with CD36. Because TSP-1 but not TGF-beta1 is a natural ligand for CD36, these findings suggest that the L-TGF-beta1 in a complex with TSP-1 localizes to the macrophage cell surface when TSP-1 interacts with its receptor, CD36. Furthermore, the association of TSP-1/L-TGF-beta1 complex with CD36 is necessary to the activation of L-TGF-beta1 because antibodies to CD36 prevent the colocalization of TGF-beta1 with CD36 as observed by immunofluorescence and inhibit activation of the L-TGF-beta1 by explanted alveolar macrophages. These findings suggest that activation of L-TGF-beta1 by plasmin occurs at the cell surface of activated alveolar macrophages and requires a TSP-1/CD36 interaction.