恶性疟原虫抗原对健康人外周血T细胞免疫功能的影响

目的 目的 探讨恶性疟原虫抗原对健康人外周血T淋巴细胞免疫功能的影响。方法 方法 健康成人外周血单个核细胞 （PBMC） 体外分别用恶性疟原虫 （P. f） 抗原 （5 μg/ml） 和正常红细胞 （nRBC） 抗原 （5 μg/ml） 进行刺激, 同时给予IL?2维持细 胞增殖, 另外设立只加IL?2的阴性对照组进行培养。培养12 d时, 刺激组细胞再用对应的抗原刺激20 h, 用流式细胞仪 检测T淋巴细胞亚群分泌IL?4和IFN?γ的情况, 并用羧基荧光素乙酰乙酸琥珀酰亚胺酯 （Carboxyfluorescein diacetate, suc? cinimidyl ester, CFSE） 标记法检测T细胞增殖反应。结果 结果 健康人PBMC经P. f抗原刺激扩增后CD4+ T细胞的增殖指数 （PI） 明显高于nRBC抗原刺激组和阴性对照组 （P均＜0.05）, 但3组γδ T的PI差异无统计学意义 （P＞0.05）。P. f抗原组 分泌IL?4的CD4+ T细胞百分率明显高于nRBC抗原组和阴性对照组 （P均＜0.05）, 但3组分泌IFN?γ的CD4+ T细胞百分 率差异无统计学意义 （P＞0.05）。结论 结论 P. f抗原在体外可刺激健康人外周血CD4+ T细胞增殖活化, 后者通过优先分泌 IL?4而发挥免疫调节作用。     

Further studies on growth factor production by the TC-1 stromal cell line: pre-B stimulating activity.

Adherent murine stromal cells support long-term in vitro lymphopoiesis or myelopoiesis dependent on the culture conditions used. A cell line, TC-1, isolated from long-term liquid murine marrow cultures under conditions approaching those permissive for lymphoid growth, has been found to produce an activity that acts synergistically with interleukin-3 (IL-3) or colony-stimulating factor-1 (CSF-1) to stimulate in vitro myeloid colonies, but which has no intrinsic colony-stimulating activity. We report here the presence of multiple growth factors in conditioned medium (CM) from the TC-1 line, including granulocyte-macrophage colony-stimulating factor (GM-CSF) (bioassay with antibody blocking and messenger RNA [mRNA] analysis), granulocyte CSF (G-CSF) and IL-4 (factor-dependent cell line bioassay), and CSF-1 (radioimmunoassay, mRNA) along with a pre-B cell inducing activity, which appears separate from these CSFs and segregates with the myeloid synergizing activity through anion exchange, sizing, and Conconavalin A chromatography. Because these activities are not yet purified to homogeneity, their identity or lack of identity remains an open question. Assays of TC-1 CM or cellular mRNA analysis have given negative results for IL-1, IL-2, IL-3, IL-6, and IL-7, and IL-6 does not stimulate pre-B cells in this assay. However, IL-4 and G-CSF do stimulate in vitro induction of pre-B cells from pre-B and B-cell-depleted Balb/C marrow and are present in CM by selective cell line assay. A monoclonal antibody to IL-4 that inhibited its pre-B inducing activity did not inhibit pre-B inducing activity of TC-1 CM. These data suggest the existence of a unique synergizing and pre-B inducing factor(s) in TC-1 CM. Given the known capacity of subliminal levels of growth factors to act synergistically, an alternate possibility is that these biologic phenomena represent the actions of low concentrations of growth factors acting synergistically and possibly associated with some core protein.     

Induction of pre-B cell proliferation after de novo synthesis of the pre-B cell receptor

The assembly of a pre-B cell receptor (pre-BCR) composed of an Ig mu heavy chain (mu H-chain), the surrogate light (SL) chain, and the Ig alpha/beta dimer is critical for late pro-B cells to advance to the pre-B cell stage. By using a transgenic mouse model, in which mu H-chain synthesis is solely driven by a tetracycline-controlled transactivator, we show that de novo synthesis of mu H-chain in transgenic pro-B cells not only induces differentiation but also proliferation. This positive effect of mu H-chain synthesis on proliferation requires the presence of SL chain and costimulatory signals provided by stromal cells or IL-7. We conclude that pre-BCR signaling induces clonal expansion of early pre-B cells.     

Increasing the intracellular Na+ concentration induces differentiation in a pre-B lymphocyte cell line.

The ability of lipopolysaccharide (LPS), a polyclonal B-cell mitogen, to induce differentiation in the pre-B cell tumor line 70Z/3 can be mimicked by drugs that increase the intracellular Na+ concentration. Pharmacologically increasing the cellular Na+ content with monensin, a sodium ionophore, or ouabain, a specific inhibitor of Na+, K+-ATPase, induces surface IgM expression in these cells. We have shown that LPS stimulates uptake of Na+ through an amiloride-sensitive pathway. These results show that the essential action of LPS to induce surface IgM expression is activation of a Na+ uptake system.     

kappa gene diversity among the clonal progeny of pre-B lymphocytes.

Some clonal pre-B cell lines, when transformed by Abelson murine leukemia virus, are able to rearrange and express kappa light chain genes. We have analyzed the light chains expressed in sets of early B-cell subclones derived from two pre-B cell clones. Each subclone makes an indistinguishable mu heavy chain, while the kappa gene rearrangements and proteins synthesized were distinct. All members of one set of subclones expressed a V kappa 21 kappa light chain. Only one of the members of the other two sets of subclones expressed V kappa 21. Thus, a single pre-B-cell clone can select a kappa variable region from more than one family. In each subclone of the set that expressed V kappa 21 light chain the same member appears to be used. The differences detected in the expressed proteins can best be explained by primary sequence alterations in the rearranged V kappa 21 segment. These sequence alterations have resulted in a restriction site polymorphism in the expressed V kappa 21 gene and charge and size differences in the expressed proteins. These data suggest that diversification of kappa light chains can occur at the pre-B- to early B-cell stage of development.     

T-cell-depleted allogeneic bone marrow transplantation for acute leukaemia using Campath-1 antibodies and post-transplant administration of donor's peripheral blood lymphocytes for prevention of relapse

One hundred and forty-six patients with acute leukaemia (81 with ANLL and 65 with ALL) received allogeneic bone marrow transplantation from their fully matched siblings. 121 patients underwent T-cell depletion (TCD) using Campath 1 monoclonal rat anti-human lymphocyte (CDw52) antibodies; 67 with Campath 1M and 54 with Campath 1G isotypes. Patients were conditioned for transplant using either total body irradiation combined with chemotherapy (125 patients) or busulfan and cyclophosphamide (21 patients). 112 recipients of T-cell depleted allografts received in addition total lymphoid irradiation (TLI) for prevention of rejection. Engraftment of neutrophils (>0.5 × 10⁹/l) and platelets (>25 × 10⁹/l) occurred on days 15 and 18, and on days 18 and 20 in recipients of Campath 1M and Campath 1G treated marrows respectively. Rejection was documented in 6.8% of T-cell depleted transplants. Leukaemia relapse-free survival at 2 years was 83% for patients transplanted in first CR, 76% in second CR (P²= 0.34) and 42% in advanced leukaemia (P²= 0.009). 81 marrow recipients, 38 with Campath 1M and 43 with Campath 1G treated marrow, received post-transplant graded increments of donor's peripheral blood lymphocytes (PBL) to induce graft-versus-leukaemia (GVL) effects. Administration of donor's PBL was associated with clinically significant GVHD and with decreased relapse rate especially in patients with ALL. Our data suggest that in patients receiving marrow allografts depleted of T cells by Campath 1 monoclonal antibodies, rejection can be reduced by adequate pregrafting immunosuppression. In patients with advanced disease, post-transplant cell-mediated immunotherapy (CMI) using donor's PBL may be beneficial; however, further studies are needed to define the optimal schedule of CMI for safe and effective prevention of relapse following TCD bone marrow transplantation in malignant haematological diseases.     

Fibronectin promotes cell proliferation of human pre-B cell line via its interactions with VLA-4 and VLA-5

Abstract

Fibronectin (FN) is thought to play an important role in various aspects of hematopoiesis through binding to very late antigen (VLA)-4 and VLA-5. Little is known, however, about the effects of FN on the proliferation of B cell progenitors. In this study, we investigated the effects of immobilized FN on the proliferation of the pre-B cell line, Nalm-6, which expresses both VLA-4 and VLA-5. Immobilized FN significantly promoted the proliferation of Nalm-6 cells through the synergistic effects of VLA-4 and VLA-5. Furthermore, FN induced the phosphorylation of mitogen-activated protein kinases (MAPKs) of Nalm-6 cells. The MAPK kinase 1 (MEK1) inhibitor, PD98059, and Src family tyrosine kinase inhibitor, herbimycin A, inhibited the FN-promoted proliferation of Nalm-6 cells. These results demonstrate that the interactions of FN and VLA-4/VLA-5 transmit the growth signals that are mediated through Src family tyrosine kinases and the MAPK cascade in Nalm-6 cells. The precise mechanism of synergistic effect of VLA-4 and VLA-5 on FN-promoted proliferation of Nalm-6 cells should be further investigated.     

Fibronectin promotes cell proliferation of human pre-B cell line via its interactions with VLA-4 and VLA-5

Fibronectin (FN) is thought to play an important role in various aspects of hematopoiesis through binding to very late antigen (VLA)-4 and VLA-5. Little is known, however, about the effects of FN on the proliferation of B cell progenitors. In this study, we investigated the effects of immobilized FN on the proliferation of the pre-B cell line, Nalm-6, which expresses both VLA-4 and VLA-5. Immobilized FN significantly promoted the proliferation of Nalm-6 cells through the synergistic effects of VLA-4 and VLA-5. Furthermore, FN induced the phosphorylation of mitogen-activated protein kinases (MAPKs) of Nalm-6 cells. The MAPK kinase 1 (MEK1) inhibitor, PD98059, and Src family tyrosine kinase inhibitor, herbimycin A, inhibited the FN-promoted proliferation of Nalm-6 cells. These results demonstrate that the interactions of FN and VLA-4/VLA-5 transmit the growth signals that are mediated through Src family tyrosine kinases and the MAPK cascade in Nalm-6 cells. The precise mechanism of synergistic effect of VLA-4 and VLA-5 on FN-promoted proliferation of Nalm-6 cells should be further investigated.     

Reproducible production of protective human monoclonal antibodies by fusion of peripheral blood lymphocytes with a mouse myeloma cell line

The production of monoclonal antibodies of human origin may represent a significant advance in immunotherapy for disease in humans. Although human monoclonal antibody has been produced from human lymphocytes by fusion with human myeloma cell lines or by Epstein-Barr viral transformation, fusion of postimmunization human lymphocytes with a mouse myeloma cell line is a relatively simple and reproducible alternative. Mouse-human hybrid cell lines were obtained in 205 (53%) of the microtiter wells initially seeded. Thirty-one (15%) of these hybrid cell lines secreted antibody of predefined specificity. Cloning was attempted with eight of the hybrid cell lines, and long-term antibody production was established in four of the lines: two hybridomas secreted antibody to the capsule of Haemophilus influenzae type b, one secreted antibody to tetanus toxoid, and one secreted antibody to diphtheria toxin. The production of mouse-human hybridomas appears to be a reliable method for obtaining human monoclonal antibody of predefined specificity.     

H1 histone subtype constitution and phosphorylation state of the ageing cell system of human peripheral blood lymphocytes

Until a few years ago, the H1 histones were exclusively considered to be the architectural proteins of chromatin involved in chromatin condensation. However there is now increasing data to support the hypothesis that the H1 subtypes are involved in genomic integrity and that they may have unexpected functional roles in various biological processes such as in differentiation and DNA repair, apoptosis and lifespan. Moreover, the H1 histones are phosphorylated to a great extent. Recent work has implicated phosphorylation of H1 in the regulation of chromatin remodeling. In light of the fact that chromatin reorganization and heterochromatin formation has been shown to take place during ageing and senescence, in the present investigation, we have analyzed the changes that take place in the somatic H1 linker histone subtype profile and their phosphorylation states in human peripheral blood lymphocytes as a function of donor age. Results from this work show that there is a significant age-related dephosphorylation of H1.4 and H1.5 and an increase in the heterochromatin protein HP1alpha as a function of donor age. These results indicate that dephosphorylation of H1 histones may be related to an increase in senescence-associated heterochromatin formation during the in vivo ageing of human peripheral blood lymphocytes.     

Toxic-shock-syndrome toxin 1-induced proliferation of lymphocytes: comparison of the mitogenic response of human, murine, and rabbit lymphocytes

Toxic-shock-syndrome toxin 1 (TSST 1) produced by Staphylococcus aureus induced in vitro proliferation of lymphocytes isolated from rabbit spleens, murine spleens, and both human peripheral blood and cord blood. This mitogenic response was nonspecific in all three systems. In the mouse and human systems, proliferation depended upon the presence of macrophages in the responding lymphocyte population. Inability to remove macrophages from rabbit splenocyte suspensions made it impossible to determine the contribution of this cell in rabbit splenocyte proliferation. Kinetic analysis of TSST 1-induced mitogenicity showed that proliferation of lymphocytes was maximal between days 4 and 6 in each of the three systems examined. Sensitivity to TSST 1 was similar in each system, with maximal proliferation achieved at TSST 1 doses as low as 0.1 ng/5 X 10(5) murine splenocytes or 2 X 10(5) rabbit splenocytes, and 0.01 ng/3 X 10(5) human mononuclear cells from human peripheral blood. Study of separated populations of mouse splenocytes and mononuclear cells from human peripheral blood showed that the TSST 1-induced proliferative response resided solely in the T cell populations.     

Galectin-1 is a stromal cell ligand of the pre-B cell receptor (BCR) implicated in synapse formation between pre-B and stromal cells and in pre-BCR triggering

Although preB cell-receptor (pre-BCR) formation and cell-surface expression is essential for B cell development, pre-BCR generation of signal transduction remains elusive. Here, we report that recombinant pre-BCRs and the surrogate light chain bind specifically to the bone marrow stromal cell galectin-1 (GAL1), an S-type lectin. The surrogate light chain/GAL1 association is a direct protein-protein interaction (K(a) = 2 x 10(6) M(-1)), and the NH(2) extra loop of lambda-like is the major binding element. Pre-BCR binding to stromal cells depends upon GAL1 anchoring to glycosylated counter-receptors, and these complexes completely relocalize to form a synapse at the contact zone between preB and stromal cells. This immune developmental synapse is accompanied by the initiation of intracellular tyrosine kinase activity and signal transduction from the pre-BCR.     

Avenins from different cultivars of oats elicit response by coeliac peripheral lymphocytes

OBJECTIVE: The avoidance of oats in coeliac patients is still controversial. If oats is confirmed to be safe, it would be a valuable component and offer more variation in a gluten-free diet. The aim of this work was to evaluate whether avenins from different varieties of oats show different abilities in the activation of coeliac peripheral lymphocytes. MATERIAL AND METHODS: In order to assess whether the immunogenic effect of oats varies according to the cultivar, peripheral lymphocytes from 10 coeliac children were exposed to avenins from four different oats varieties: Lampton, Astra, Ava and Nave. Lymphocyte proliferation and interferon-gamma (IFN-gamma) release in the culture medium were measured as indexes of immune activation. RESULTS: All the varieties of oats tested were immunogenic, with Lampton and Ava avenins inducing lymphocyte activation similar to that activated by wheat gliadin, while Astra and Nave avenins showed less immunogenicity, but still with a measurable effect. CONCLUSIONS: There are still concerns about the suitability of including oats in a gluten-free diet. Coeliac patients consuming oats-containing food should be carefully monitored, until there is more evidence to show the safety of oats and varieties of low-toxicity oats.     

Avenins from different cultivars of oats elicit response by coeliac peripheral lymphocytes

Objective. The avoidance of oats in coeliac patients is still controversial. If oats is confirmed to be safe, it would be a valuable component and offer more variation in a gluten-free diet. The aim of this work was to evaluate whether avenins from different varieties of oats show different abilities in the activation of coeliac peripheral lymphocytes. Material and methods. In order to assess whether the immunogenic effect of oats varies according to the cultivar, peripheral lymphocytes from 10 coeliac children were exposed to avenins from four different oats varieties: Lampton, Astra, Ava and Nave. Lymphocyte proliferation and interferon-gamma (IFN-γ) release in the culture medium were measured as indexes of immune activation. Results. All the varieties of oats tested were immunogenic, with Lampton and Ava avenins inducing lymphocyte activation similar to that activated by wheat gliadin, while Astra and Nave avenins showed less immunogenicity, but still with a measurable effect. Conclusions. There are still concerns about the suitability of including oats in a gluten-free diet. Coeliac patients consuming oats-containing food should be carefully monitored, until there is more evidence to show the safety of oats and varieties of low-toxicity oats.     

Clonal expansion of limited T cell clonotypes in affected muscle from a patient with post-transplant polymyositis

Critical roles of T cells in idiopathic polymyositis have been suggested, but, those in polymyositis occurring as GVHD after BMT are poorly understood. We thus investigated T cell clonality in a patient with post- transplant polymyositis. As a result, T cell receptor beta chains used various BV families in peripheral blood, but only one BV family (BV7) in affected muscle. Importantly, T cells proliferated oligoclonally both in the peripheral blood and the muscle, however, the expanded clonotypes were completely different. Taken together, T cells expanded in the muscle, possibly stimulated by limited kinds of antigens, may drive myositis.     

Monoclonal antibodies against the human leukemia cell line K 562

Three monoclonal antibodies raised against K 562, a cell line originally established from a patient with chronic myeloid leukemia (CML) in terminal blast crisis, were selected according to their distinct reaction pattern. Whereas two antibodies (ZIK-C1-A/C5 and ZIK-C1-A/H5 also designated C and H) recognized antigens, present on K 562 cells and other immature and mature hematopoietic cells (cell lines and normal blood and bone marrow cells), antibody ZIK-C1-A/D9 also designated Y showed an exclusive binding to K 562 cells. The results obtained (here and in the following paper) indicate, that antibody ZIK-C1-A/D9 defines an early differentiation antigen of hematopoiesis or a leukemia-associated antigen.     

Sustained decrease of peripheral lymphocytes after allogeneic blood stem cell aphereses

48 healthy donors underwent peripheral blood stem cell (PBSC) apheresis for allogeneic transplantation beginning on day 4 of G-CSF (2 × 5 μg/kg) mobilization. In one to four (median two) large-volume mononuclear cell aphereses, a median of 55.9 × 10⁹ of lymphocytes (range 21.0–109.2 × 10⁹) were collected, an amount comparable to lymphocyte numbers removed by therapeutic lymphaphereses in autoimmune diseases. Mean peripheral lymphocyte counts decreased from premobilization values of 2.31 × 10⁹/l to 1.31 × 10⁹/l at a median of 34 d (1 month) and 1.53 × 10⁹/l at a median of 327 d (11 months). The decrease in peripheral lymphocyte counts was significantly correlated with the number of lymphocytes removed and the number of aphereses. Neutrophil and platelet counts returned to normal values after 1 month whereas monocyte counts and haemoglobin concentrations were significantly decreased at 1 month but not at 11 months.     

VLA-4 is involved in the engraftment of the human pre-B acute lymphoblastic leukaemia cell line NALM-6 in SCID mice

Attachment of human pre-B leukaemic cells to human or murine bone marrow stromal cells in vitro is largely mediated by the β₁ integrin VLA-4 binding to VCAM-1. Cells subsequently migrate within the stroma, a process also involving VLA-4. A variant of the pre-B acute lymphoblastic leukaemia cell line NALM-6, designated 4A1, lacking expression of VLA-4, was generated by radiation-induced mutagenesis followed by several rounds of negative selection with immunomagnetic beads, fluorescence activated cell sorting and clonal expansion. In vitro assays using 4A1 cells showed reduced binding to, and migration under, the murine stromal line M2-10B4. Sublethally irradiated mice (n = 19) with severe combined immunodeficiency were injected intravenously with NALM-6 cells. Animals developed signs of leukaemia with hind-limb paralysis at a median of 30 d (95% confidence interval 28–30). Although there were no gross abnormal findings at autopsy, histological analysis revealed extensive marrow replacement and focal liver infiltration with leukaemic blasts, which were confirmed to be of human origin by flow cytometry. 12 mice were injected with a similar number of cells from the VLA-4-negative variant cell line 4A1. Six mice developed signs of leukaemia after 43–74 d, with the remaining six being free of signs of disease after > 100 d (P < 0.001). Mice in this group with leukaemia had a lower incidence of hind-limb paralysis and less leukaemic infiltration in the marrow, but in some cases had large tumour nodules elsewhere. After a single 500 μg intraperitoneal injection of anti-murine VCAM-1 monoclonal antibody (MK2.7), five additional mice were injected with an identical number of wild-type (VLA-4⁺) NALM-6. All animals developed signs of leukaemia after a similar period to those injected with wild-type NALM-6 only. These results demonstrate that the β₁ integrin VLA-4 is involved in the engraftment of the pre-B-cell leukaemic cell line NALM-6 in SCID mice, although the interaction with VCAM-1 is unlikely to be the sole explanation.