Rosette formation between human lymphocytes and sheep erythrocytes. Inhibition of rosette formation by specific glycopeptides.

Rosette formation with unsensitized sheep erythrocytes is a characteristic of human thymus dependent lymphocytes. Release of glycopeptides from the sheep erythrocyte by trypsin reduces rosette formation. These tryptic glycopeptides inhibit rosette formation by untrypsinized sheep erythrocytes; this suggests that rosetting is mediated by erythrocyte surface glycopeptides. To investigate the molecular nature of this interaction, we examined the abilities of various model compounds to act as haptenic inhibitors of rosette formation. Inhibition is given by glycopeptides bearing oligosaccharide units rich in sialic acid, galactose, N-acetylglucosamine, and mannose linked to asparagine residues through glycosylamine bonds. Among compounds tested, fetuin glycopeptide is most effective, but human transferrin glycopeptide and human erythrocyte glycopeptide I also inhibit rosette formation. Other compounds including human erythrocyte glycopeptide II, human IgG glycopeptide, lacto-N-neotetraose, 3'- and 6'-sialyllactose show no significant inhibition. Neither sialic acid, galactose, manose, nor N-acetyl-glucosamine alone inhibits rosette formation. Stepwise degradation of fetuin glycopeptide established the galactose residues as important determinants of inhibitory activity. Fetuin glycopeptide blocks rosette formation when added to a suspension of human lymphocytes and sheep erythrocytes or when preincubated with human lymphocytes, but not when preincubated with sheep erythrocytes. Studies of the binding of [3H] fetuin glycopeptide to normal lymphocytes demonstrate 7.5 x 10(6) saturable binding sites per cell. No saturable binding of this compound to sheep erythrocyte membranes is observed. Compared to normals, lymphocytes from patients with chronic lymphatic leukemia demonstrate decreased fetuin glycopeptide binding with a mean of 0.9 x 10(6) sites per cell. This decreased binding correlates with the impaired ability of these cells to form rosettes. The data suggest that fetuin glycopeptide inhibits rosette formation by binding to the thymus-dependent cell where competition occurs with sheep erythrocytes for specific lymphocyte surface receptors.     

Purification of soluble human T lymphocyte receptors for sheep erythrocytes.

Using a polyclonal heterologous anti-soluble E-receptor serum, we identified molecules of molecular weight circa 58,000 and 150,000. The soluble receptor molecule with molecular weight of approximately 58,000 (Rs1) was initially purified from supernatant of heated lymphocytes through chromatography on Sephadex G-200 and/or DEAE-cellulose. The soluble receptor molecule with molecular weight of approximately 150,000 (Rs2) is detected at high levels in the serum of patients with cancer and uremia. Rs1 and Rs2 present in serum from cancer patients were purified by chromatography on Sephadex G-200 and by affinity chromatography using anti-Rs1 IgG. 131I-labelled supernatant of heated lymphocytes binds to sheep erythrocytes and the elution and analysis of the molecules adsorbed showed bands of molecular weights approximately 58,000 and 150,000, confirming the receptor activity of these molecules.     

Human autologous mixed lymphocyte reactivity is primarily specific for xenoprotein determinants adsorbed to antigen-presenting cells during rosette formation with sheep erythrocytes

We present evidence that most T cells proliferating in response to autologous sheep erythrocyte (SRBC)-separated non-T cells (NT) cells are not specific for autoantigens but for antigens derived from xenogeneic sources. The conclusion was based on the following three observations. First, we found that NT cells isolated in the absence of xenoproteins by means of density gradient centrifugation on Percoll only weakly stimulated autologous T cells. Because this weak proliferation could not be expanded in restimulation experiments, its significance as an immune recognitive event remains questionable. NT cells isolated by the above method in the absence of xenogeneic determinants readily acquired stimulatory capacity after brief exposure to either SRBC or fetal calf serum. Second, restimulation of T memory cells generated in 1 degree autologous mixed lymphocyte reaction (AMLR) against SRBC-separated autologous NT cells was exclusively seen when NT cells exposed to or separated with xenoproteins were used for restimulation. Third, T memory cells generated against SRBC-separated autologous NT cells were specifically restimulated by autologous Percoll-separated NT cells that had been pulsed with a variety of xenogeneic mammalian sera. These xenogeneic determinants were preferentially recognized in context with autologous HLA-DR+ cells. From these findings and from our previous results that indicated an absolute requirement of HLA-DR+-adherent NT cells (8), we conclude that human AMLR primarily does not represent an autoantigen but a xenoantigen response that is genetically restricted by the HLA-DR type of the antigen-presenting cell.     

Molecular forms of soluble human T lymphocyte receptor for sheep erythrocytes in serum and saliva.

Using a specific serum anti-soluble T lymphocytes receptor for sheep erythrocytes (E) and SDS-PAGE, we detected radioactive bands of molecular weight 58,000 in immunoprecipitates of supernatant of heated human lymphocytes (SHL), in the supernatant of PHA stimulated lymphocyte cultures (SLC), normal human serum (NHS), and serum from cancer and uremia patients, labelled with 131I. By Sephadex G-200 chromatography, in addition to this fraction, we detected molecules of molecular weight higher than 150,000 which interact with the anti-soluble receptor serum (anti-RS), in serum from cancer and uremia patients. These molecules were detected in NHS or SHL after concentration or by prolonged exposure of SDS-PAGE with some labelled and immunoprecipitated SHL samples. The soluble receptors of molecular weights 58,000 (RS1) and more than 150,000 (RS2) were fully identical when analyzed by immunodiffusion with anti-RS serum. When submitted to immunoelectrophoresis, RS1 showed electrophoretic migration similar to that of albumin, while RS2 showed a pattern close to that of alpha 2-globulin. However, RS2 did not show antigenic relationship with IgM and was not an immune complex with IgG. Even though the presence of RS in human saliva has not yet been reported, molecules that interact with anti-RS serum have been detected in human saliva and are fully identical to molecules found in supernatant of heated human T lymphocytes and NHS. The RS molecules present in human saliva have a molecular weight and electrophoretic migration similar to those of RS1 from SLC and from human serum and have no antigenic relationship with human albumin.     

Effects of human interleukin 1 and human tumor necrosis factor on human T lymphocyte colony formation.

The growth of T lymphocyte colonies in agar is dependent on the cooperation of a number of different cell types and their products. Among these interacting cells are monocytes and/or macrophages. Monocytes are capable of producing both interleukin 1 (IL-1) and tumor necrosis factor (TNF). These two factors display a positive influence on T cell colony formation. Recombinant IL-1 and recombinant TNF were both shown to stimulate T cell colony growth in a dose-dependent manner, and this stimulation could be blocked by prior incubation with anti-IL-1 or anti-TNF, respectively. In addition, conditioned medium obtained from monocytes cultured in the presence of endotoxin also stimulated T cell colony formation. This stimulation by monocyte-conditioned medium was partially suppressed by incubation with anti-TNF or anti-IL-1, while incubation with both antibodies together displayed greater suppression. In conclusion, monocytes produce at least two factors, IL-1 and TNF, which can stimulate T cell colony formation by peripheral blood lymphocytes.     

淋巴细胞分离液对T细胞阳性花环和非T细胞阳性花环计数结果影响的研究

目的 探讨单克隆抗体SPA红细胞花环法(McAb-A-E法)检验T淋巴细胞亚群时,淋巴细胞分离液对阳性花环细胞计数结果以及非T淋巴细胞阳性花环计数结果的影响.方法 选择淋巴细胞分离液法分离的单个核细胞(PBMC)和不加任何物质的抗凝血用自然沉降法分离的混合细胞,用10倍量稀释液洗涤1次.两种细胞用McAb-A-E直接法...     

Quantification of the soluble receptor of human T lymphocytes for sheep erythrocytes in the serum of patients with aplastic anaemia

The soluble E-receptor (Rs) of human T lymphocytes for sheep erythrocytes has been quantified by electroimmunodiffusion ("rocket" electrophoresis) in serum samples from 23 patients with aplastic anaemia and 43 controls. A statistically significant increase of Rs levels was found in the group of patients as compared with controls. Sixteen patients showed Rs values greater than the mean +2SD of the control group. No correlation between Rs values and the number of circulating total lymphocytes or T lymphocytes was observed. Since Rs serum levels have previously been shown to be increased in diseases associated with depressed cell mediated immunity, the finding of high levels of Rs in most cases of aplastic anaemia may contribute to the understanding of the pathogenesis of this disease.     

Mechanisms responsible for defective human T-lymphocyte sheep erythrocyte rosette function associated with hepatitis B virus infections.

The expression of selected lymphocyte surface-membrane markers was evaluated in 37 patients with acute viral hepatitis B, 10 of whom were studied serially through the resolving and convalescent phases of disease. Bone marrow-derived (B) lymphocytes were identified by reference to surface immunoglobulin, whereas normal thymus-derived (T) lymphocytes were assayed by their capacity to form spontaneous nonimmune rosettes with sheep erythrocytes (E rosettes, ER). During the acute and resolving phases of viral hepatitis B, the relative and absolute number of ER-positive lymphocytes was significantly reduced, whereas the number of surface immunoglobulin-positive lymphocytes and the absolute lymphocyte count remained normal. This resulted in the appearance of a third population of cells, deficient in respect to both surface immunoglobulin and ER markers. Such cells accounted for nearly 25% of peripheral blood lymphocytes, approximately 5 x 105ml blood. Depression of the number of ER-positive lymphocytes occurred at least once during the course of disease in every patient studied serially, and was observed in 55 of 67 individual assays of the 37 cases of acute viral hepatitis B. Lymphocytes from some patients reacquired ER function when cultured in fetal calf serum but not in the presence of autologous serum. Such autologous serum was capable of suppressing ER function of lymphocytes from normal donors. The extrinsic suppression of er function by a serum factor (designated as the Rosette Inhibitory Factor), was found to be time dependent, characterized by a 4-h latent period and requiring approximately 18 h for maximum attenuation of ER function. The Serum Rosette Inhibitory Factor was: (a) heat and freeze-thaw stable, (b) nondialyzable, (c) physically separable from hepatitis B surface antigen, (d) not a lymphocytotoxic antibody, and (e) had the buoyant density of a lipoprotein. This extrinsic mechanism was observed in 41.8% of patients with reduced numbers of ER-positive lymphocytes. The Rosette Inhibitory Factor was not detectable in the serum of the remaining 58.2% of the cases of acute and resolving viral hepatitis B despite the presence of reduced numbers of ER-positive lymphocytes. The lymphocytes from these cases did not reacquire ER function when cultured in the absence of autologous serum. The mechanisms responsible for the suppression of normal ER function in these cases appears to be intrinsic to the lymphocytes and not the result of a humoral factor. The T lymphocyte lineage of cells deficient in respect to ER function, whether of intrinsic or extrinsic type, was demonstrated by their capacity to form spontaneous rosettes with neuraminidase-treated sheep erythrocytes. Both intrinsic and extrinsic suppression of T lymphocyte ER function commonly occurred during the first 4 wk of acute viral hepatitis B.9 of the 10 patients followed serially continued to manifest defective ER function at 12 wk...     

麻疹患者T淋巴细胞亚群和调节性T淋巴细胞分析

目的 探讨成年人及儿童麻疹患者急性期与恢复期T淋巴细胞及调节性T淋巴细胞表达水平的变化。 方法 成年人及儿童麻疹患者各40例,用流式细胞术分别测定各组出疹期和恢复期患者外周血CD+3总T细胞、CD+4辅助性T细胞(Th)、CD+8细胞毒性T细胞(Tc)及CD+4CD+25调节性T淋巴细胞(Treg)比例。 结果 成年人麻疹患者出疹期较对照期外周血CD+3总T和CD+4Th表达水平降低,CD+4CD+25 Treg表达水平升高,恢复期CD+4Th升高,CD+8Tc降低,CD+4/CD+8比值升高,CD+4CD+25 Treg降低。儿童麻疹患者出疹期较对照组CD+3总T、CD+4Th降低,恢复期较出疹期CD+3总T表达回升。 结论 T淋巴细胞及调节性T淋巴细胞参与成年人及儿童麻疹患者免疫状态的调节,但其表达水平在成年人和儿童中有差异。     

Phenomenon of human T cells rosetting with sheep erythrocytes analyzed with monoclonal antibodies. “Modulation” of a partially hidden epitope determining the conditions of interaction between T cells and erythrocytes

Anti-D66 is a monoclonal antibody able to inhibit E-rosette formation of T cells both at 4 degrees C and at 37 degree C but that does not inhibit T cell rosette formation with neuraminidase or 2-amino-ethylisothiouronium bromide (AET)-pretreated E. As demonstrated by capping experiments, it defines an epitope, D66, that is directly involved in E-rosette formation. D66 is distinct from the epitope defined by 9.6 because 9.6, a previously defined “pan-T” monoclonal antibody, inhibits E(AET) rosette formation and because no cross-blocking occurred between both antibodies fixation. However, 9.6 and D66 are carried by the same molecule, as demonstrated by sequential immunoprecipitation assays performed on two different T cell lines. On the thymocyte surface, also, 9.6 and D66 are most probably carried by the same molecule, as indicated by cocapping and colysostripping experiments. D66 is present at higher densities on thymocytes and activated T cells than on peripheral blood T cells. Investigation of numerous T cell populations, both normal and malignant, showed a straightforward correlation between elevated D66 density and ability to form 37 degrees C stable E-rosettes. Neuraminidase treatment of thymocytes and peripheral blood lymphocytes forming E-rosettes unmasked a large fraction of D66 not readily accessible on their surface. These hidden D66 epitopes appear to be responsible for a surprising observation: the ability of anti-D66 to inhibit E-rosette formation could be totally reversed by fixation on anti-D66 of an antibody to mouse immunoglobulin or an Fab fragment anti-mouse immunoglobulin. This would induce microdisplacement with emergence of hidden D66, as documented by fluorometric studies. Finally, malignant T cells with a differentiative status of mature T cells, but forming no (or low numbers of) E-rosettes, could be induced both to display D66 and to form E-rosettes by neuraminidase treatment.     

Antigen-binding T cells as helper cells. Separation of helper cells by immune rosette formation

The spleen T cells from mice immunized 6 days earlier with either chicken gamma globulin (CGG) or with donkey erythrocytes (DRC) were rosetted with CGG-coated sheep erythrocytes or with DRC. The immune rosettes (RFC) (antigen-binding cells) were separated from the bulk of nonrosette-forming cells (non-RFC) by 1-g velocity sedimentation and the RFC and non-RFC tested for helper activity in cooperative antihapten responses in vitro. RFC or non-RFC were mixed with normal or hapten-primed spleen cells, challenged with the appropriate hapten-carrier conjugate and cultured for 4 days in Marbrook tissue cultures. The helping activity was quantitated from the numbers of antihapten antibody-producing cells generated per culture. The results show that specific helper cell activity could be selectively recovered in the immune rosette-forming cell population whereas the non-RFC population was depleted of help. These findings indicate that the helper T cells express specific antigen binding receptors.     

Human T lymphocyte "E" rosette function. I. A process modulated by intracellular cyclic AMP

The capacity of normal human T lymphocytes to form rosettes with sheep red blood cells can be inhibited by drugs or agents which induce elevations in intracellular levels of cyclic AMP. The effect is early in the presence of agents which elicit rapid elevations in intracellular cyclic AMP (isoproterenol, aminophylline) and occurs later in the presence of cholera toxin which induces a dalayed increase in endogenous cyclic AMP. Dibutyryl cyclic AMP is inhibitory, and the effects of dibutyryl cyclic AMP and the adenyl cyclase stimulators are potentiated by inhibition of phosphodiesterase. These data provide substantial evidence that elevation of intracellular cyclic AMP diminishes E rosette function of lymphocytes.     

T淋巴细胞及其亚群与运动免疫

已有大量的文献资料表明T淋巴细胞及其亚群和人体运动免疫调节有非常重要的联系。T淋巴细胞是一种重要的免疫活性细胞,是机体免疫细胞中数目最多,作用最重要的功能细胞。T淋巴细胞亚群中的CD4细胞和CD8细胞亚群是重要的免疫调节细胞,可增强或抑制其他免疫细胞的活性。对T淋巴细胞及其亚群与运动免疫的关系,急性运动,有氧运动和长期适量运动对T淋巴细胞及其亚群的免疫功能的影响,以及T淋巴细胞及其亚群在运动免疫中作用的机制和相关影响因素进行了论述。     

Idiotype-like determinants on human T lymphocytes alloactivated in mixed lymphocyte culture

T cells alloactivated in 5-d MLC with an HLA-DR-different stimulator acquire the capacity of stimulating the autologous mixed lymphocyte response (AMLR). We have demonstrated that activation of AMLR by allosensitized T cells is determined by the expression of the idiotype receptor for the stimulating HLA-DR alloantigen. This has been shown in experiments in which purified, OKT-3-positive T cell suspensions were first primed for 9 d with AMLR-activated T lymphoblasts, then tested in secondary AMLR with autologous lymphoblasts sensitized to various HLA- DR alloantigens. Accelerated memory responses were induced only by autologous lymphoblasts that had been sensitized against the same HLA- DR specificity as the primary AMLR stimulators. This response was not inhibited by a mouse monoclonal antibody recognizing Ia-like determinants, and was not triggered by human allogeneic resting peripheral blood lymphocytes. Thus, recognition of alloactivated T lymphoblasts in secondary AMLR seems to be specific for the idiotype- like determinants expressed by the autologous stimulators.     

Herpes simplex virus (HSV) antigens on HSV-transformed rat cells can be demonstrated by rosette formation with Staphylococcus aureus protein-A-coated sheep red blood cells

Two rat embryo cell lines transformed by herpes simplex virus (HSV) and four tumor cell lines derived from inoculation of HSV-transformed cells formed rosettes after treatment with rabbit or human anti-HSV IgG and sheep red blood cells coated with protein A of Staphylococcus aureus, Cowan I strain. The frequency of rosette-forming cells after treatment with rabbit anti-HSV IgG at a concentration of 0.01 mg/10(6) cells varied between 41.6 and 73% depending on the cell line. Pretreatment of HSV-transformed cells with rabbit anti-HSV IgG Fab prevented rosette formation after treatment with rabbit or human anti-HSV IgG. Trypsin digestion of transformed cells reduced significantly the proportion of rosettes formed with human or rabbit anti-HSV IgG. The microscopic visualization of rosette formation on HSV-transformed cells is consistent with the expression of HSV-specific antigens on the cell surface.