Two distinct types of murine blast colony-forming cells are multipotential hematopoietic precursors

Two types of blast colonies can be stimulated to develop in semisolid agar cultures of murine bone marrow cells. Typically, these are either multicentric colonies stimulated by stem cell factor (SCF) plus interleukin-6 (IL-6) or dispersed colonies stimulated by Flt3 ligand (FL) plus IL-6. Both types of blast colony-forming cells (BL-CFCs) can generate large numbers of lineage-committed granulocyte-macrophage progenitor cells and exhibit some capacity for self-generation and the formation of eosinophil and megakaryocyte progenitor cells. However, the two populations of BL-CFCs are largely distinct and partially separable by fluorescence-activated cell sorting and are distinguished by differing capacity to form granulocyte-committed progeny. Both types of BL-CFCs can generate dendritic cells and small numbers of lymphocytes but the FL-responsive BL-CFCs have a greater capacity to form both B and T lymphocytes. Both types of blast colonies offer remarkable opportunities to analyze multilineage commitment at a clonal level in vitro.     

Morphological variants of leukemic cells in B chronic lymphocytic leukemia are associated with different T cell and NK cell abnormalities

B chronic lymphocytic leukemia (B-CLL) is a heterogeneous disease. The different morphological variants of leukemic B cells appear to define different clinical groups of patients. Several abnormalities have been found in T lymphocytes and natural killer (NK) cells from B-CLL patients. We have investigated the phenotypic and functional characteristics of purified CD2+ cells from B-CLL patients at Binet's stage A and classified according to the neoplastic B lymphocyte morphology criteria: 32 patients with typical B-CLL and 12 patients with atypical B-CLL. Forty-three age and sex matched healthy controls were also studied. In fresh purified CD2+ cells from typical B-CLL patients, percentages of CD4+, CD4+CD45RA+, CD8+CD45RA+ T lymphocytes and CD3−CD56+ (NK) cells were significantly higher than those found in atypical B-CLL patients. However, in DC2+ cells from typical B-CLL patients, percentages of CD3+, CD3+DR+, CD8+, CD4+CD45RO+, and CD3+CD56+ cells were significantly lower than those found in atypical B-CLL patients. Increased percentage of NK cells was only found in typical B-CLL patients. The proliferative response and the production of interleukin (IL)-2 and IL-4 by phytohemagglutinin (PHA) stimulated CD2+ cells were significantly higher in typical B-CLL patients than in atypical B-CLL patients. We concluded that different patterns of phenotypic and functional alterations in the T lymphocytes and NK cells of B-CLL patients are found in patients with typical or atypical B-CLL defined according to the morphology of the leukemic cells. Am. J. Hematol. 55:175–182, 1997. © 1997 Wiley-Liss, Inc.     

51Cr Labelling of Normal Human T and B Lymphocytes for Kinetic Studies in Vivo

Lymphocyte traffic between blood and tissues was assessed by ⁵¹Cr labelling of lymphocytes and subsequent autologous reinfusion in 10 normal elderly persons. The technique for isolation and platelet depletion of lymphocyte suspensions is described. By the labelling procedure used about 70 μCi ⁵¹Cr may be incubated in about 100 million lymphocytes. This permits measurement of lymphocyte-bound radioactivity on the T and B fractions separately. The blood disappearance curves for labelled lymphocytes indicate the existence of exchangeable pools in the tissues of T as well as of B lymphocytes, that of the T lymphocytes being apparently larger. A characteristic finding in the blood disappearance curves for total lymphocytes is an increase in lymphocyte-bound radioactivity in the blood 4–6 h after reinfusion, designated reappearance. The disappearance curve of the B lymphocytes shows reappearance 4–10 h after reinfusion, whereas that of the T lymphocytes falls exponentially without any recordable reappearance. On the basis of the disappearance curves and a knowledge of the topographic distribution of T and B lymphocytes in the lymphoid tissues, a model of T and B lymphocyte traffic in the lymph nodes is discussed. This model operates with T and B lymphocyte passage by way of postcapillary venules and describes the migration in and around the germinal centres. The T lymphocytes in the periphery of the germinal centres are assumed to derive mainly from the afferent lymph, whereas the B lymphocytes in the centres are exchanged with lymphocytes in the blood in an exchangeable pool. The functional implications are discussed.     

A low blood lymphocyte count is associated with an expansion of activated cytotoxic lymphocytes in patients with B-cell chronic lymphocytic leukaemia

Abstract: In order to determine the relationships between CD2+ lymphocyte subpopulations and tumour mass, the immunophenotype of natural killer (NK) cells and T lymphocyte subsets was studied in 56 B-chronic lymphocytic leukaemia (B-CLL) patients and 38 healthy subjects. The patients were classified according to their blood lymphocyte count (BLC). Forty patients had BLC<30×10⁹/l (low BLC, less tumour mass) and 16 patients had BLC>30×10⁹/l (high BLC, larger tumour mass). The percentage of CD3^– CD56+ cells, as well as of CD8+, CD8+CD45RO+ and CD3+CD57+ T subsets in low BLC patients, were higher than those found in high BLC patients. Conversely, the percentages of CD3+HLA DR+, CD4+ and CD4+CD45RO+ lymphocytes were higher in high BLC patients than in low BLC patients. The CD4/CD8 ratio was decreased in low BLC patients while it was increased in high BLC patients and a significant positive correlation was found between their CD4/CD8 ratio and their BLC. We conclude that in low BLC B-CLL patients there is a decreased percentage of activated helper lymphocytes and an increased percentage of NK cells and activated cytotoxic T lymphocytes. These results suggest a role for NK cells, and helper and cytotoxic T lymphocytes in the control of tumour burden in B-CLL patients.     

Traffic of T and B Lymphocytes in the Normal Spleen

The occurrence of C3b and C3d complement receptors and of Fc and E receptors in normal splenic tissue was studied in cryostat sections incubated with specific marker cells. Thereby the topographic distribution of T and B lymphocytes in the splenic tissue was mapped: T^E lymphocytes are found especially in the periarteriolar sheaths, B lymphocytes in eccentric zones around the arterioles. The cells in the splenic pulp carry especially Fc, but also C3b receptors, whereas C3d receptors appear to be specific of lymphocytes. The basic lymphocyte traffic behind the mapped distribution of the lymphocyte subpopulations in the spleen was elucidated by lymphocyte kinetic studies using autologous ⁵¹Cr-labelled reinfused lymphocytes. These studies were performed partly on normal persons and partly on splenectomized persons without immune defects. The results indicate the presence of an exchangeable pool of T as well as of B lymphocytes in the spleen, the T cell pool being larger and making up in normals about 30 % of the total exchangeable T lymphocyte mass. The matrix for the T lymphocyte pool in the spleen is the periarteriolar sheaths. The difference in structure between the lymph nodes and the lymphoid tissue of the spleen, including differences in the distribution of lymphocyte subpopulations, are explained by the special vascular arrangement in the spleen.     

Standardization of a technique for analysing the frequency of parasite-specific cytotoxic T lymphocyte precursors in cattle immunized with Theileria parva

Summary A limiting dilution microculture system was optimized to quantify the frequency of Theileria parva-specific cytotoxic T lymphocyte precursors (CTLp) in peripheral blood mononuclear cells (PBMC) from immune cattle. Optimal results were obtained with responder cell input levels ranging from 2 × 10⁴/well to 6.25 × 10²/well, along with 1–5 × 10³/well stimulator cells in standard supplemented RPMI 1640 medium containing 2.5–5% T cell growth factors. Thirty-six microtitre wells were established at each responder input level. Cultures were incubated for 7 days at 38°C, at the end of which time individual wells were screened for cytotoxic activity in a 4-h ¹¹¹indium oxine-release assay. Analysis of the cytotoxicity data, by a computer-programmed maximum likelihood estimation method indicated that they conformed to the Poisson model of single-hit kinetics. Estimates of frequencies ranged from 1:3600 to 1:5275 CTLp in PBMC of eight cattle between 1 and 24 months after immunization with T. parva. By contrast, no CTLp were detected in six naïve animals analysed to a responder cell input of 10⁵/well. Split-well analysis of individual microwells showed that the CTL clones generated under limiting dilution conditions displayed exquisite specificity for parasitized cells, were genetically restricted and in some animals were parasite strain-specific.     

A Colchicine-Sensitivity Test for Leukaemic Lymphocytes

Non-dividing lymphocytes from patients with chronic lymphocytic leukaemia were more sensitive than normal lymphocytes to reagents as prednisolone, cytarabine, vincristine and colchicine. The maximum difference was obtained when the cells were incubated with colchicine at 37°C for 20 h. The sensitivity was measured by a ‘sensitivity index’ which was an estimate of the average percentage of lymphocytes killed by 1.0 and 0.1 μg/ml of colchicine. The index was 0–1% for lymphocytes from the blood of 14 normal persons and was 61–98% for 23 of 25 leukaemic patients with absolute lymphocyte counts of 8,000 times 10⁹/1 or more. 3 of 4 untreated patients with presumptive diagnoses of early leukaemia had low absolute counts of 3,300 to 7,600 times 10⁹ lymphocytes/I and high sensitivity indices of 41 to 83%. Tests on treated patients with lymphocyte counts less than 8,000 times 10⁹/1 suggesed a correlation of the index with remission and relapse. Hairy cells from 3 patients with hairy cell leukaemia were resistant to colchicine. Sensitivity to colchicine seemed useful at a test for leukaemic lymphocytes and as an aid in the haematologic evaluation of patients with chronic lymphocytic leukaemia and malignant lymphoma.     

Ultrastructural localization of acid phosphatase in immunologically defined neoplastic lymphocytic cells and hairy cells A comparison between two different substrates

The subcellular localization of acid phosphatase (AcP) in various immunologically-defined neoplastic lymphoid cells including hairy cells was investigated by electron microscopy. 2 substrates, naphthol-AS-BI-phosphoric acid (naphthol-AS-BI-P) and sodium β-glycerophosphate, were compared. By incubation in naphthol-AS-BI-P containing medium, the reaction product was found located in granules, vesicles, the Golgi apparatus, the rough ER including the nuclear envelope in the cells of T ALL, T CLL and HCL. A typical pattern of reaction was observed for each of these disorders: enzyme-positive Golgi membranes and neighbouring granules, clustered in the nuclear notch in T cell-derived lymphoblasts; enzyme-positive granules around Gall bodies, aggregated paranuclearly in T CLL lymphocytes and enzyme-positive scattered cytoplasmic granules and vesicles in hairy cells. Enzyme activity was occasionally seen in singly-occurring granules in the cells of cALL, B CLL and B PLL, rarely in other substructures. With the Gomori method using β-glycerophosphate as substrate, the enzyme reaction was limited primarily to lysosomal sites and was seldom observed in other organelles. Tartrate-resistant AcP was found in the majority of hairy cells and in a few prolymphocytes, located in the same structures as AcP without tartrate.     

Blood B and T Lymphocytes and In Vitro Cellular Immune Reactivity in Untreated Human Malignant Lymphomas and Other Malignant Tumors

Peripheral blood lymphocytes and their in vitro reactivity have been recorded prior to treatment in 18 patients with Hodgkin's disease, 11 with lymphosarcoma, 13 with reticulosarcoma, 20 with various solid tumors and 37 normal control persons. The mean total numbers of lymphocytes, those of T lymphocytes, and the mean reactivities to PHA and ConA were reduced in all groups except lymphosarcoma, although with varying degrees of statistical significance. The percentages of T and B lymphocytes appeared to be normal in all groups, but the ranges of values were somewhat greater than among the normal controls. The mean total numbers of B lymphocytes were normal in all patient groups. All reductions seemed to be more pronounced in patients with disseminated than in those with localized disease, but none of these differences was statistically significant. All patient groups appeared to have reduced reactivity in MLC, while the ability to stimulate control lymphocytes was nearly normal. The results fail to indicate any in vitro immunological abberation specific to Hodgkin's disease. It seems that human malignant, neoplastic diseases are associated with a relatively selective reduction of the total numbers and reactivity of blood T lymphocytes. Various explanations of the reactivity impairment are proposed. The pathogenesis of the reduction of the total number of blood T lymphocytes remains obscure.     

Blast crisis supervening on chronic lymphocytic leukaemia. A monoclonal progression of the disease as defined by cell surface markers

Acute leukaemia is a rare event during the course of chronic lymphocytic leukaemia (CLL), and only a small fraction of such cases have been shown to be true acute lymphoblastic crises. 1 case is described where both small lymphocytes and proliferating lymphoblasts have the same monoclonal pattern as defined by direct immunofluorescence of membrane-bound immunoglobulins. Previous cases are reviewed and do not appear to be mere coincidence: acute blast crisis may represent a part of the natural history of CLL.     

一种乙型肝炎病毒转录后调节基序的体外合成方法

目的 探索T7 RNA聚合酶体外转录方法制备乙型肝炎病毒（HBV）转录后调节基序（HPRE）。方法 用聚合酶链反应法从含HBV全基因组的模板中扩增HBV HPRE基因，重组人pGEM—11zf载体，并用T7 RNA聚合酶对其进行体外转录。结果 成功地构建含HPRE基因的重组质粒，并成功地用体外转录方法制备了高纯度的HPRE。结论 T7 RNA聚合酶体外转录方法可用于制备高纯度的HPRE，为以后的深入研究奠定了基础。     

Analysis of blood T-cell cytokine expression in B-chronic lymphocytic leukaemia: evidence for increased levels of cytoplasmic IL-4 in resting and activated CD8 T cells

The cytoplasmic cytokines of purified blood T cells (CD4/C]D8) in B-CLL patients ( n  = 5) and controls ( n  =5) were evaluated by flow cytometry. The mean levels of cytoplasmic IL-4 were significantly elevated in resting and activated B-CLL CD8 cells compared to control CD8 cells. IL-4 cytoplasmic levels were comparable for resting B-CLL and control CD4 cells but greater for B-CLL activated CD4 cells. The mean fluorescence intensity of B-CLL CD8 cytoplasmic IL-4 was 4–5-fold greater, indicating higher IL-4 density per CLL CD8 than control CD8 cells. Both CLL CD4 and CD8 cells post-activation had higher levels of cells double positive for cytoplasmic IL-4 and interferon. These data indicate that freshly isolated CD8 and CD4 blood T cells from B-CLL patients have significantly elevated (above control) levels of commitment to expression of IL-4. Since IL-4 has an important modulatory impact on CLL and normal B cells, this observation has implications regarding the biology of B-CLL.     

Chronic lymphocytic leukemia: A monoclonal disease

A black woman with chronic lymphocytic leukemia (CLL) was found to have monoclonal B lymphocytes with one type of surface immunoglobulin and one variant of G6PD (glucose-6-phosphate dehydrogenase) (G6PD A). Erythrocytes and T cells contained both G6PD A and G6PD B and hence were of polyclonal origin. The CLL cells in this patient likely arose from a developmental stage later than the step of differentiation into T and B lymphocytes. Furthermore, her erythrocytes did not arise from a stem cell affected by the CLL process.     

Characterization of tumour cells in concomitant chronic lymphocytic leukaemia and acute monocytic leukaemia.

A 68-year-old woman presented with haematological changes of chronic lymphocytic leukaemia and acute monocytic leukaemia. This diagnosis was confirmed by identification of cell surface markers for T and B lymphocytes and the identification of abnormal immunoglobulins and lysozyme in serum and urine.     

Activation of peripheral blood lymphocytes in patients with primary sjögren's syndrome

Summary The peripheral blood from 15 patients with primary Sjögren's syndrome and from 15 matched controls was examined for the presence of activated T and B lymphocytes, by using monoclonal antibodies directed to interleukin-2 (IL-2) and transferrin receptors, and to HLA-DR determinants. The number of circulating positive-T cells was significantly greater in the patients than in the controls, irrespective of disease activity. There were more of the CD8 cells than of the CD4 cells that expressed IL-2 receptors. There was a small but significant increase in activated B cells in the patients, since this population is virtually absent from the normal blood.     

Lymphocyte subpopulations in insulin-dependent diabetics with and without serum islet-cell autoantibodies

Summary The lymphocyte subpopulations of 26 insulin-dependent diabetics were studied. Thirteen of them had persistent pancreatic islet-cell serum antibodies (ICA) (mean (±SD) duration of diabetes 11 ± 8 years). The others were ICA-negative (mean duration of diabetes 10±8 years). The mean fasting blood glucose in the week before the lymphocyte count was 1.37±.45 g/1 (two specimens for every patient). As controls 19 healthy volunteers, sex and age matched, were investigated. The T-lymphocyte count was no different in diabetics compared to controls, B-cells were significantly raised (p <0.01) in the ICA-positive group, when tested with antihuman gammaglobulin sera (IgG + IgA + IgM), anti-human IgG and anti-human IgM, while no difference was observed between ICA-negative patients and normal subjects. IgA-bearing lymphocytes were equally raised in both diabetic groups (p <0.05). These data show an altered immunological balance in type IB (autoimmune) diabetes, characterized by an increased number of B-lymphocytes.     

慢性乙型肝炎患者树突状细胞的培养鉴定和功能特点

目的：研究慢性乙型肝炎患者树突状细胞（DC）的功能特点,探讨其与肝炎发病机制的关系。方法：分离获得18例慢性乙型肝炎患者和10例正常人外周血单个核细胞（PBMC）,在体外培养条件下,加入细胞因子重组人粒细胞巨噬细胞集落刺激因子（GM－CSF）,IL－4,胎肝酪氨酸激酶受体的配体（FLt3-L)和肿瘤坏死因子α（TNFα）,使DC细胞增殖,成熟,用流式细胞仪检测DC的表面标志,同时检测DC在混合淋巴细胞反应（MLR）中的刺激能力和细胞因子的分泌。结果：DC在体外经细胞因子的刺激可明显增殖,但慢性乙型肝炎患者DC的增殖速度低于正常人;DC表面标志人组织相容性复合物Ⅱ类分子（HLA－DR）,CD－80（B7－1）,CD－86（B7－2）和CD－1α的表达较正常对照组均明显降低（P＜0．001）,尤以CD－1α的降低更为显著,DC在MLR中的刺激能力亦明显低于正常对照。并且与正常对照相比,其产生的IL－12水平降低,而NO水平却增高（P＜0．05）。结论：慢性乙型肝炎患者DC的表型不成熟和功能的缺失,由此导致IL－12的产生和刺激T细胞增殖能力的降低,可能是HBV感染持续发展的原因之一。     

Distribution of B,T, and O Lymphocytes in Blood and Tissues of Normal Humans Reflecting a Kinetic Model

The occurrence of the lymphocyte subpopulations T^E, B^Fo, B^C3, B^{Fc + C3}, B^Ig, and O^-, percentage as well as absolute, was studied in lymphocyte suspensions from tonsils, abdominal lymph nodes, spleens, bone-marrow aspirates, and at the same time in venous blood. The absolute lymphocyte content (number of lymphocytes per g tissue) was highest in the abdominal lymph nodes, lower in the spleens and tonsils, and lowest in the blood. T^E lymphocytes were found in the significantly highest percentage, 60%, in the blood. B lymphocytes, comprising B^Fc+C3 plus B^Ig, were present in the highest percentage in the bone marrow: 74%. Tonsils, spleens, and abdominal lymph nodes contained fewer B lymphocytes, and the blood fewest: 39%. A significant correlation was found only between the absolute numbers of T and B lymphocytes. A relationship between the absolute number of T lymphocytes and the total number of B lymphocytes as well as fractions thereof was thus demonstrated in the various tissues and in the blood and also between the blood and the tissue. O^- lymphocytes were found in bone marrow, lymph nodes, and spleens, apparently as markerless precursors of other subpopulations. The main conclusion of the study is: In the lymphocytokinetic system the T lymphocytes must play a guiding role as an afferent vector, trigging the B fractions which thus constitute the efferent vector of the system.     

HBsAg、HBcAg活化树突状细胞治疗慢性乙型肝炎的体外研究

目的 研究慢性乙型肝炎患者外周血树突状细胞(DC)经HBsAg、HBcAg活化后的免疫功能.方法 从慢性乙型肝炎患者外周血中培养扩增DC,在DC成熟前,加入纯的HBsAg、HBcAg刺激,用流式细胞仪检测DC表型,用液闪计数仪观察DC对T细胞的增殖作用,用ELISA法检测混合淋巴细胞反应(MLR)中细胞因子的分泌水平.结果 经HBcAg刺激DC的CD86表达率为(92.14±5.12)%,明显高于HBsAg刺激组和未加抗原组(P＜0.01);经HBcAg刺激组DC诱导同种异体静止T细胞增殖的能力每分钟液闪计数值(cpm)为34259±3127,明显高于HBsAg刺激组(20258±2917)和单个核细胞组(3469±417),P＜0.01;经HBcAg刺激组DC MLR中IL-12浓度为(342±42.3)ng/L,分别高于HBsAg刺激组和未加抗原组(P＜0.01).结论 体外经HBcAg刺激DC可有效提呈抗原病毒,并可进一步刺激T细胞产生.