B细胞活化基因的研究Ⅰ．人活化B细胞cDNA文库的构建

本文报导以人活化Ｂ淋巴细胞的３Ｄ５细胞的ｍＲＮＡ为模板，逆转录合成ｃＤＮＡ的第一链：按常规的自身引物法，以合成的第一链为模板合成ｃＤＮＡ第、二链；以及λｇｔ１１Ｓｆｉ－Ｎｏｔ噬菌体为载体，定向插入构建了人活化Ｂ细胞。ＤＮＡ表达文库。该文库的大小为２．５×１０６ｐｆｕ，插入片段长度大于１ｋｂ；用抗ＣＤ７１及ＣＤ９８单克隆抗体为探针，对所构建的文库进行筛选，均获得了阳性克隆，ＣＤ７１及ＣＤ９８均为人Ｂ细胞活化时才得以表达的分化抗原，从而证明，新构建的ｃＤＮＡ文库是人活化Ｂ细胞的ｃＤＮＡ表达文库。     

Yersinia Enterocolitica O:3 Isolated from Patients with or without Reactive Arthritis Induces Polyclonal Activation of B Cells and Autoantibody Production In Vivo

The mechanisms by which arthritis-provoking pathogens such as Yersinia enterocolitica interact with the human immune system to produce inflammatory synovitis are not well known. One of the immunomodulating mechanisms used against these pathogens is the polyclonal activation of lymphocytes. In this study, we investigated the extent of the B-lymphocyte activation induced in mice by a strain of Y. enterocolitica O:3 (FCF 526) isolated from a patient with arthritis, and compared it with two other strains, a virulent one (FCF 397[+]) isolated from a patient without arthritis and its plasmidless isogenic pair (FCF397[-]). Also we investigated the production of autoantibodies in mice infected with these different strains. SPF Swiss mice were infected intravenously with a suspension of Y. enterocolitica. Spleen cells were taken on days 7, 14, 21 and 28 after infection and the number of cells secreting nonspecific and specific antibodies of IgG¹, IgG^2a, IgG^2b, IgG³, IgM and IgA isotypes were determined by the ELISPOT technique. The presence of autoantibodies in mouse serum was investigated by the dot-blot assay. The pattern of infection of the three bacterial strains were almost the same. We observed a general increase in the number of nonspecific Ig-secreting cells with all three strains, and the greatest increases observed were in the IgG^2a and IgG³ isotypes. Only a small fraction of the immunoglobulins detected were antibacterial, suggesting that the rest resulted from polyclonal B cell activation. The strain isolated from the patient with arthritis (FCF526) induced the greatest production of autoantibodies, coinciding with the period in which the greatest activation of nonspecific B lymphocytes was seen. There were no signs of arthritis or inflammation in the joints of the infected animals. Based on our results, we were unable to determine whether there is an association between the arthritogenic capability of Y. enterocolitica and polyclonal activation of B cells.     

双特异抗体体外模拟特异B细胞抗原递呈作用

用化学偶联的双特异性抗体复合物－αＴＴ－α－ＩｇＭ通过体外实验，观察其抗原递呈能力。结果显示：αＴＴ－αＩｇＭ的对照组低１０００倍左右，与抗原特异性Ｂ细胞的抗原递呈效应相似。抑制试验显示：羊抗鼠ＩｇＭ，鼠ＩｇＭ能抑制αＴＴ－αＩｇＭ对非特异性Ｂ细胞抗原递呈作用的增强效应，表明Ｂ细胞膜ＩｇＭ在抗原递呈中起关键性作用。本研究结果表明：αＴＴ－αＩｇＭ的存在确能模拟抗原特异性Ｂ细胞的高效抗原递呈功能。     

Phenotypic characterization of inflammatory cells from osteoarthritic synovium and synovial fluids

Osteoarthritis (OA) is considered a degenerative joint disorder caused by mechanical wear to the articular surface. However, while joint injury, obesity, and mutations in collagen increase the risk of developing OA, evidence implicates inflammatory mechanisms in disease progression and chronicity. To address this question we used FACS analysis, immunohistochemistry, and in vitro cell culture to evaluate inflammatory mechanisms in synovial fluids and joint tissues obtained after arthrocentesis or knee replacement surgery. Immunohistochemistry revealed a significant T cell infiltrate in six of nine tissue specimens. T cells were present throughout the synovial membrane and were particularly localized around vasculature and in large cellular aggregates. Cells within the aggregates expressed markers associated with immune activation and antigen presentation. T cells from OA synovial fluids expressed an activated phenotype and synthesized interferon-gamma following in vitro stimulation. These data support the hypothesis that inflammatory cells play a significant role in OA disease progression and chronicity.     

Activation of genetically major histocompatibility complex (MHC) class II-deficient B lymphocytes

It has been suggested that MHC class II molecules can transduce signals required for B-cell activation. Enhancement or inhibition of B-cell stimulation by anti-MHC class II molecule antibodies has likewise been reported. The study of B cells from patients with a combined immune deficiency due to a defective expression of MHC class II genes provides a useful tool for approaching the functional role of B-cell HLA class II molecules. We have thus analyzed the specific and nonspecific, cognate and noncognate B-cell activation of genetically HLA class II-deficient lymphocytes. B lymphocytes from 14 tested patients were able to synthesize RNA following stimulation with ionomycin and phorbol myristate acetate or anti- antibodies and with mannan, a T cell-independent polysaccharidic antigen. They were also able to synthetize DNA following the addition of ionomycin and PMA or of anti- antibodies in the presence of recombinant interleukin 2. Pokeweed mitogen failed to induce B-lymphocyte terminal differentiation into immunoglobulin-producing cells in the presence of normal T lymphocytes, while a combination of anti-CD2 antibodies were capable of triggering IgG synthesis. B-cell activation, whatever the condition used, did not induce HLA class II expression. Mannan-specific T cell-dependent antibody production (IgM) was detected in 6 of 14 patients. Anti-influenza virus antibody production was always found absent. These results are compatible with the hypothesis that B-cell activation events that do not require a cognate interaction with T cells can occur in the absence of HLA class II molecule expression, while the absence of HLA class II molecule expression prevents T-B cognate interaction.     

Potassium Humate Inhibits Complement Activation and the Production of Inflammatory Cytokines <Emphasis Type="Italic">In Vitro</Emphasis>

The effects of brown coal derived potassium humate on lymphocyte proliferation, cytokine production and complement activation were investigated in vitro. Potassium humate increased lymphocyte proliferation of phytohaemaglutinin A (PHA) and pokeweed mitogen (PWM) stimulated mononuclear lymphocytes (MNL) in vitro from concentrations of 20 to 80 μg/ml, in a dose dependant manner. On the other hand potassium humate, at 40 μg/ml, significantly inhibited the release of TNF-α, IL-1β, IL-6 and IL-10 by PHA stimulated MNL. Regarding complement activation it was found that potassium humate inhibits the activation of both the alternative and classical pathways without affecting the stability of the red blood cell membranes. These results indicate that the anti-inflammatory potential of potassium humate could be partially due to the inhibition of pro-inflammatory cytokines responsible for the initiation of these reactions as well as inhibition of complement activation. The increased lymphocyte proliferation observed, might be due to increased IL-2 production as previously been documented.     

Ex Vivo Programming of Antigen-Presenting B Lymphocytes: Considerations on DNA Uptake and Cell Activation

Plasmids used in DNA vaccination not only serve as a source of antigen, but also have an important adjuvant effect. This review focuses on recent advancements made in understanding how cells internalize DNA, and how internalized DNA activates immune response pathways. We also comment on the role of B cells in both of these processes.     

Aberrant B Cell Selection and Activation in Systemic Lupus Erythematosus

The detrimental role of B lymphocytes in systemic lupus erythematosus (SLE) is evident from the high levels of pathogenic antinuclear autoantibodies (ANAs) found in SLE patients. Affirming this causative role, additional antibody-independent roles of B cells in SLE were appreciated. In recent years, many defects in B cell selection and activation have been identified in murine lupus models and SLE patients that explain the increased emergence and persistence of autoreactive B cells and their lowered activation threshold. Therefore, clinical trials with B cell depletion regimens in SLE patients were initiated but disappointingly the efficacy of B cell depleting agents proved to be limited. Remarkably however, a major breakthrough in SLE therapy was accomplished by blocking B cell survival factors rather then eliminating B cells. This surprising finding indicates that although SLE is a B cell-driven disease, the amplifying crosstalk between B cells and other cells of the immune system likely evokes the observed tolerance breakdown in B cells. Moreover, this implies that intelligent interception of pro-inflammatory loops rather then selectively silencing B cells will be key to the development of new SLE therapies. In this review, we will not only highlight the intrinsic B cell defects that facilitate the persistence of autoreactive B cells and their activation, but in addition we will focus on B cell extrinsic signals derived from T cells and innate immune cells that lower the activation threshold for B cells.     

B Cell Response and Hemagglutinin Stalk-Reactive Antibody Production in Different Age Cohorts following 2009 H1N1 Influenza Virus Vaccination

The 2009 pandemic H1N1 (pH1N1) influenza virus carried a swine-origin hemagglutinin (HA) that was closely related to the HAs of pre-1947 H1N1 viruses but highly divergent from the HAs of recently circulating H1N1 strains. Consequently, prior exposure to pH1N1-like viruses was mostly limited to individuals over the age of about 60 years. We related age and associated differences in immune history to the B cell response to an inactivated monovalent pH1N1 vaccine given intramuscularly to subjects in three age cohorts: 18 to 32 years, 60 to 69 years, and ≥70 years. The day 0 pH1N1-specific hemagglutination inhibition (HAI) and microneutralization (MN) titers were generally higher in the older cohorts, consistent with greater prevaccination exposure to pH1N1-like viruses. Most subjects in each cohort responded well to vaccination, with early formation of circulating virus-specific antibody (Ab)-secreting cells and ≥4-fold increases in HAI and MN titers. However, the response was strongest in the 18- to 32-year cohort. Circulating levels of HA stalk-reactive Abs were increased after vaccination, especially in the 18- to 32-year cohort, raising the possibility of elevated levels of cross-reactive neutralizing Abs. In the young cohort, an increase in MN activity against the seasonal influenza virus A/Brisbane/59/07 after vaccination was generally associated with an increase in the anti-Brisbane/59/07 HAI titer, suggesting an effect mediated primarily by HA head-reactive rather than stalk-reactive Abs. Our findings support recent proposals that immunization with a relatively novel HA favors the induction of Abs against conserved epitopes. They also emphasize the need to clarify how the level of circulating stalk-reactive Abs relates to resistance to influenza.     

Mechanisms and implications of adaptive immune responses after traumatic spinal cord injury

Traumatic spinal cord injury (SCI) in mammals causes widespread glial activation and recruitment to the CNS of innate (e.g. neutrophils, monocytes) and adaptive (e.g. T and B lymphocytes) immune cells. To date, most studies have sought to understand or manipulate the post-traumatic functions of astrocytes, microglia, neutrophils or monocytes. Significantly less is known about the consequences of SCI-induced lymphocyte activation. Yet, emerging data suggest that T and B cells are activated by SCI and play significant roles in shaping post-traumatic inflammation and downstream cascades of neurodegeneration and repair. Here, we provide neurobiologists with a timely review of the mechanisms and implications of SCI-induced lymphocyte activation, including a discussion of different experimental strategies that have been designed to manipulate lymphocyte function for therapeutic gain.     

Activation of human T cells through CD6: functional effects of a novel anti-CD6 monoclonal antibody and definition of four epitopes of the CD6 glycoprotein

The CD6 glycoprotein is expressed primarily on lymphocytes andconveys co-activating signals to T cells, but its exact functionand ligand(s) are unknown. A novel mAb, termed UMCD6, was demonstratedto recognize CD6 by immunoprecipitation, Western blotting, andreactivity with COS cells transfected with CD6 cDNA. UMCD6 wasmitogenic for T cells and was strongly synergistic with phorbolester in inducing T cell activation. UMCD6 enhanced the autologousmixed lymphocyte reaction as previously observed with anotheranti-CD6 mAb, anti-T12. The activating effects of UMCD6 weremore striking than those of other anti-CD6 mAbs and encompassedall of the diverse stimulatory properties previously reportedfor other anti-CD6 reagents. However, neither UMCD6 nor otheranti-CD6 antibodies alone or in combination with phorbol esteror IL-2 were able to induce thymocytes to proliferate. Stimulationby UMCD6 is dependent on accessory cell function in a mannernot accounted for simply by antibody crosslinking. UMCD6 didnot induce an increase in cytoplasmic free Ca²⁺, but the CD6activation pathway appears to involve protein kinase C. UMCD6and a panel of seven other anti-CD6 mAbs were used in a seriesof experiments to define four discrete epitopes of CD6 usingthe criteria of antibody cross-blocking, reactivity on reducedWestern blots, and resistance to controlled V8 protease digestion.The functional mAbs UMCD6, 2H1, and antl-T12 each recognizeda different epitope. Taken together, the results of these studiesstrongly reinforce the hypothesis that CD6 plays a significantand distinct role in T cell activation, and suggest that multipleregions of CD6 may be functionally active.     

Activation of cytokines corroborate with development of inflammation and autoimmunity in thromboangiitis obliterans patients

Thromboangiitis obliterans (TAO) is a segmental inflammatory occlusive disorder that affects the arm and leg arteries of young smokers. The immune system seems to play a critical role in the aetiology of TAO; however, knowledge of the aspects involved in the progression of vascular tissue inflammation and, consequently, the evolution of this disease is still limited. This study was carried out to investigate the cytokine levels of tumour necrosis factor (TNF)‐α, interleukin (IL)‐1β, IL‐4, IL‐17 and IL‐23 in the plasma of TAO patients presenting with acute clinical manifestations. The study included 20 TAO patients (n = 10 women; n = 10 men) aged 38–59 years under clinical follow‐up, classified into two groups: (i) TAO former smokers (n = 11) and (ii) TAO active smokers (n = 9); the control groups included normal volunteer non‐smokers (n = 10, active smokers (n = 10) and former smokers (n = 10). Patients' plasma samples were measured using the sandwich enzyme‐linked immunosorbent assay. Statistical analyses were performed using the non‐parametric Mann–Whitney U‐test, with parameters significant at P < 0·05. The activities of all cytokines were different in groups of TAO patients when compared with normal controls, and decreased for control smokers. Increased levels of TNF‐α, IL‐1β, IL‐4, IL‐17 and IL‐23 were significant in patients with TAO when compared to the controls (P < 0·005, all parameters). The results presented here indicate an increased production of cytokines in TAO, possibly contributing to the inflammatory response observed in the patients' vascular levels. In addition, the increased levels of IL‐17 and IL‐23 suggest that the disturbance of TAO is involved with mechanisms of autoimmunity. Thus, the discovery of IL‐17 and its association with inflammation and autoimmune pathology has reshaped our viewpoint regarding the pathogenesis of TAO, which was based previously on the T helper type 1 (Th1)–Th2 paradigm.     

Activation and re-activation potential of T cells responding to staphylococcal enterotoxin B

To elucidate the parameters that lead to superantigen inducednon-responsiveness, an in vitro model for studying primary andsecondary responses to the bacterial superantlgen staphylococcalenterotoxin B (SEB) was established. Upon re-activation withSEB, in vitro SEB primed T cells show an early proliferativeresponse that ‘quenches’ in time and is severelyimpaired 3 days after re-stimulatlon. Despite their overallimpaired proliferative capacity and IL-2 production, these Tcells are able to produce IFN-_ß and to up-regulateactivation markers CD69 and IL-2R upon re-stimulation with SEB,demonstrating that SEB non-responsiveness is not absolute. Rather,it reflects the inability to mount an ongoing proliferativeresponse upon re-stimulation with SEB. Our results also demonstratethat SEB-induced non-responsiveness is not simply the resultof presentation in the absence of co-stimulation, since presentationof SEB on highly purified dendritic cells during the primaryresponse did not prevent the induction of non-responsiveness.Aspreviously shown, SEB induces a T_h1 phenotype in respondingCD4⁺ T cells. Skewing towards a T_h2 phenotype by adding IL-4and antibodies to IFN-_ß did not prevent the inductionof non-responsiveness by SEB. Interestingly, T cells pretreatedwith plate-bound anti-CD3 and anti-V_ß8 were also non-responsiveto SEB re-stimulation. Thus, non-responsiveness to SEB (definedhere as inability to produce IL-2 and proliferate) seems toreflect an intrinsic inability of previously activated T cellsto respond to SEB, probably reflecting differences in signaltransduction pathways used by naive versus previously activatedT cells.     

Dendritic Cell Production of Cytokines and Responses to Cytokines

Dendritic cells (DC) are a family of bone marrow-derived MHC class II expressing cells which occur in small numbers in most lymphoid and nonlymphoid tissues. They represent a distinct lineage of leukocytes which can be found in two distinct maturational stages: immature dendritic cells are exemplified by the Langerhans cells in the epidermis, and are considered to be precursors to the mature dendritic cells in the lymphoid organs. These maturational stages can be distinguished by phenotypic and functional characteristics. Immature dendritic cells are weak stimulators of resting T lymphocytes but are excellent in processing soluble protein antigens for presentation to T cell clones. Mature dendritic cells show exactly reciprocal features. In this review the relatively few available data on cytokine production by DC and responses of DC to cytokines are collected. Our goal is to consider the role of cytokines in DC function including the transition from immature to mature stages.     

尿毒症患者透析前后外周血B细胞和T细胞及其亚群的研究

应用单克隆抗体技术检测了３５名正常人和３１例尿毒症患者外周血Ｂ细胞和Ｔ细胞亚群。结果显示：尿毒症患者外周血Ｂ细胞数显著地高于正常人（Ｐ〈０．００１）,ＣＤ３、ＣＤ４低于正常人（Ｐ〈０．０１）,ＣＤ４／ＣＤ８又非常显著地低于正常人（Ｐ〈０．００１）。因此认为,尿毒症患者为一种自身免疫调节异常的疾病。     

Activation antigen expression on human T cells. I. Analysis by two-colour flow cytometry of umbilical cord blood, adult blood and lymphoid tissue

Activation antigens (actags) were detected on T cells at low levels of intensity by carefully defining negative cells with a panel of control antibodies. The mean percentage of blood T cells from healthy volunteers that expressed actags were 22% (CD25), 54% (CD26), 38% (CD38), 12% (CD54), 6% (CD69) and 21% (HLA-DR). The variability of actag expression detected by this sensitive method was determined on healthy volunteers by repeated estimation over a year. The percentage of T cells expressing CD25 and CD26 varied no more than repeated estimation of the CD4 T cell subset, whereas other actags showed greater variability. The antigen density of these actags on T cells was determined in relation to CD4 antigen density, and for most actags ranged from 10% to 75% of the level of CD4 antigen density except for CD7 and HLA-DR, which could exceed that of CD4. Different degrees of actag expression characterized T cells from different blood and lymphoid tissues. CD26, CD38 and CD45RA were universally expressed in cord blood at higher antigen density than adult blood. This immature pattern was consistent with recent thymic emigration. CD25, CD45RO, CD54 and HLA-DR progressively increased from cord blood through adult blood to lymphoid tissues, consistent with antigen-driven activation, whereas CD26 and CD45RA decreased. CD69, a very early activation antigen, abruptly increased in lymphoid tissue, exceeding CD25 by two-to-three-fold and suggesting a pre-activation state that may not involve commitment to antigen-driven proliferation. CD7 and CD38 expression was higher in cord blood and lymphoid tissue than in adult blood, indicating both an antigen-independent and -dependent up-regulation.     

Altered Signaling Lymphocytic Activation Molecule (SLAM) Expression in HIV Infection and Redirection of HIV-Specific Responses via SLAM Triggering

Signaling lymphocytic activation molecule (SLAM) is a transmembrane lymphocytic receptor which gets rapidly upregulated following cell activation. SLAM engagement augments T cell expansion and interferon-gamma (IFN-gamma) production independently of CD28. SLAM signaling is regulated by the SLAM-associated protein. We evaluated the expression and function of SLAM on CD4(+) and CD8(+) lymphocytes in HIV-infected individuals with either recently acquired infection (Group A) or asymptomatic HIV infection (Group B) and in healthy controls (HC). Soluble antigen (HIV env peptides and tetanus toxoid)- and mitogen-stimulated proliferation and IFN-gamma and IL-10 production upon SLAM costimulation were also measured. Results showed that: (1) SLAM-expressing CD4(+) and CD8(+) lymphocytes diminish in group A patients compared to both group B patients and HC; (2) SLAM expression on CD4(+) lymphocytes is preferentially associated with the lack of CD7 on cell surface (CD4(+)CD7(-) produce IL-10 but not IFN-gamma); (3) SLAM engagement increases HIV env peptide-stimulated, but neither tetanus toxoid- nor PHA-stimulated proliferation of peripheral blood mononuclear cells (PBMC) in patients but not in HC; and (4) SLAM engagement augments IFN-gamma and reduces IL-10 production by env peptide-stimulated PBMC of HIV-infected individuals. These results demonstrate that early HIV infection results in an altered SLAM expression which correlates with a time-limited impairment of cell-mediated immunity. Furthermore, they show that triggering via SLAM potentiates HIV-specific proliferative responses with simultaneous downregulation of IL-10 and redirection of the response to TH0/TH1.