Functional characterization of mouse lymphocyte subpopulations identified by their natural binding of bacteria. I. Identification of the Ig-secreting cell subpopulation.

Three mouse B cell subpopulations (B1, B2 and B3) can be identified by their natural binding of bacteria. To determine whether these subpopulations have unique functions, we assayed the number of anti-SRBC-secreting cells and the number of Ig-secreting cells in unseparated populations and in populations in which the B2 and B3 cells were removed by immobilized monolayers of Escherichia coli-2, a bacteria that binds B2 and B3 cells. Essentially all of the plaque forming cells present in the unseparated population were found in the B1-enriched population, suggesting that most of the antibody-secreting and Ig-secreting cells were in the B1 subpopulation. To show conclusively that the anti-SRBC-secreting cells resided in the B1 subpopulation, the Jerne plaque assay was performed on slides by using lymphocytes prelabeled with various bacteria and the cells that gave rise to the plaques were directly examined. Essentially all of the secreting cells were labeled with Corynebacterium xerosis, which binds to the B1 and B2 cells, whereas very few of the secreting cells were labelled with Arizona hinshawii, which binds to the B2 cells, or with Escherichia coli-2, which binds to the B2 and B3 cells. Thus, the B1 subpopulation contained essentially all of the antibody-secreting cells, which indicates that the B cell subpopulations identified by bacteria are functionally different.     

Activation of human lymphocyte subpopulations by protein A from Staphylococcus aureus bacteria.

Cowan I strain Staphylococcus aureus bacteria were found to be mitogenic for human peripheral and cord blood lymphocytes. Experiments with lymphocyte supopulations otained by nylon wool filtration and/or E-rosette separation revealed that T-lymphocytes are the main target cells, whereas isolated B cells did not respond significantly. Further experiments suggested that B cells could be activated in the presence of mitomycin-treated T cells. Null cell-enriched lymphocyte suspensions could be stimulated by Con A but not by the bacteria or by PHA.     

Excretion of DNA by purified human lymphocyte subpopulations.

A large proportion of DNA synthesized in vitro by human lymphocytes stimulated with plant mitogens or specific antigens is selectively excreted from the cells. To determine if DNA excretion differs among various types of lymphocytes, we examined purified human lymphocyte subpopulations for DNA synthesis and excretion in response to stimulation by L-PHA. The relative proportion of newly synthesized DNA that is excreted by unseparated mononuclear cells, macrophage-depleted cells, T, and B lymphocytes is identical despite great differences in the magnitude of their responses. Low levels of both DNA synthesis and excretion by macrophage-depleted cells and B cells can be increased by reconstitution with macrophages and T cells, respectively. These data indicate that DNA exretion is a general property of lymphocytes stimulated to undergo DNA synthesis by plant mitogens.     

Alpha-fetoprotein and human lymphocyte subpopulations.

Peripheral blood lymphocytes were incubated with varying concentrations of alpha-fetoprotein and enumerated for total and "active' T lymphocytes, B lymphocytes, and their proliferative responses to phytohemagglutinin. No significant effect was observed on total T or B lymphocyte proportions. However, there was a dose-related increase in proportions of the so called "active" T lymphocytes. The response of human lymphocytes to phytohemagglutinin was markedly depressed. The alteration in the proportion of active T cells and the inhibition of T lymphocyte response to phyto hemagglutinin by alpha-fetoprotein occurred at higher concentrations than are present in amniotic fluid, serum of pregnant women, or serum of adults, but well within the range reached in fetal serum. The immunoregulatory role of alpha-fetoprotein is discussed.     

Identification of lymphocyte subpopulations with a polymethine dye

Using the polymethine dye p-ethoxyphenyl-p-aminostyryl-1,3,3-trimethyl-3H-indolium chloride as an aqueous stain applied to specimens of peripheral blood or buffy coat fixed in FAA fixative, differential coloration of leukocytes was achieved using darkfield illumination. Neutrophils stained dark maroon and contained green granules, eosinophils contained bright blue granules, basophils revealed yellow and pink granules, and monocytes stained green with green and yellow vacuoles. In studies of purified lymphocyte subpopulations obtained in a cell sorter, T-helper cells stained red, T-suppressor cells were yellow orange, B-cells appeared yellow and often contained yellow annular structures in the cytoplasm, and natural killer (NK) cells stained green and contained large green granules. As a rapid screening technique for identification of T-helper and T-suppressor cells and their ratios in health and disease, the new polymethine stain may complement the more complex monoclonal antibody techniques for identification of these cells.     

X-ray induction of micronuclei in human lymphocyte subpopulations differentiated by immunoperoxidase staining.

In this study we sought to confirm the radiosensitivity of human peripheral blood lymphocyte subpopulations using a micronucleus assay. Mononucleated cells isolated from peripheral blood were irradiated with X rays. After being cultured for 3 days, cells were fixed and stained using the immunoperoxidase staining technique. Lymphocyte subpopulations were characterized by means of the monoclonal antibodies Leu4 (CD3), Leu2a (CD8) and Leu19 (CD56). Dose-response curves were obtained by scoring the number of micronuclei in binucleated cells that reacted with a specific antibody and were then stained. The dose response of CD8+ (suppressor/cytotoxic) cells was quite similar to that of CD3+ (pan T) cells. In comparison, CD56+ (natural killer) cells were significantly less sensitive, although scorable binucleated CD56+ cells made up less than 4% of the total number of binucleated cells.     

Quantitative analysis of neutral glycosphingolipids from human lymphocyte subpopulations.

The glycosphingolipids of normal human lymphocytes from individual donors were analysed by high-pressure liquid chromatography. In addition, purified T- and B-lymphocytes were examined separately. Lactosylceramide was shown to be the major neutral glycosphingolipid in human lymphocytes, and monohexosylceramide, trihexosylceramide, globoside and paragloboside were all detected in smaller amounts. Analysis of purified B- and T-cell fractions revealed that each of these populations contained a similar qualitative profile for neutral glycosphingolipids, but that quantitatively, B-cells contained several times more of each glycosphingolipid per cell than did T-cells.     

The mixed lymphocyte reaction (MLR) stimulatory capacity of human lymphocyte subpopulations.

Using various cell separation techniques and combinations of these, suspensions were obtained highly enriched or depleted with respect to their content of E-rosette-forming T cells, Ig-bearing B cells, Fc-receptor-bearing cells, or monocytes. These purified populations were tested for their capacity to stimulate allogeneic cells in a mixed lymphocyte reaction (MLR). It could be demonstrated that the Ig-bearing B cells provide the strongest stimulus in the MLR.     

Elaboration of leukocyte migration inhibitory factor by human lymphocyte subpopulations stimulated with mitogens.

This paper describes experiments to determine whether human lymphocyte sub-populations stimulated with a variety of mitogens, leucoagglutinin (LA), concanavalin A (con A), pokeweed mitogen (PWM), protein A (prot A), and anti-β₂-microglobulin (anti-β₂m), synthesize lymphokines. T and B lymphocytes as well as unseparated mononuclear cells were stimulated with the mitogens, and the presence of leukocyte inhibitory factor (LIF) in the culture supernatants was tested by an agarose migration method. Culture supernatants stimulated with LA or prot A were also fractionated on Sephadex G-100 columns, and LIF-containing fractions were tested for heat stability and the effect of monosaccharides. The results indicated that LA and con A caused LIF synthesis only in T-cell populations, while PWM stimulated both T and B lymphocytes and prot A and anti-β₂mm were B-cell stimulants. Furthermore, LIF from LA-and prot-A-stimulated cultures behaved similarly upon physicochemical characterization.     

8株光合细菌的鉴定及其系统进化关系分析

目的为8株光合细菌(photosynthetic bacteria,PSB)作为益生菌株提供系统资料。方法用常规方法对8株PSB菌株的形态、培养特性及生理生化特征进行鉴定,同时定性分析菌株产生的类胡萝卜素和CoQ,测定菌株16S DNA序列并分析其系统进化关系,在GenBank中获取了8个16S DNA序列号。结果菌株鉴定结果表明:菌株2C、2c和13ing为沼泽红假单胞菌,Ga、Il106、WS8N为类球红细菌,MT1131为荚膜红细菌,rub为深红红螺菌。基于菌株16S DNA序列的系统进化树显示,同一种菌并不总是聚为一簇,但相隔较近;种属完全不同的菌株,尽管序列相似性高达97%以上,在系统进化树上相隔较远。结论8株PSB菌株的鉴定和系统进化关系分析结果为后续研究提供了背景资料,同时菌株在GenBank中获得的16S DNA序列号为菌株作上了生物标记,也为菌株的产权保护提供了依据。     

Binding of bacteria in lymphocyte subpopulations: role of lectin-carbohydrate interactions

Bacteria have been found to bind to lymphocyte subpopulations in a highly reproducible manner. Some of these bacteria such as B. melitensis and a strain of E. coli binds to mammalian B. cells. The binding of B. melitensis and other bacteria is due, at least in part, to lectins on lymphocytes interacting with the carbohydrates on the LPS or LTA of the bacteria. These receptors for bacteria give some indications regarding the functional potential of the cells, suggesting the possibility that the receptors identified by bacteria are used in cellular interactions with normal or malignant cells.     

Separation of human lymphocyte subpopulations. I. Transformation characteristics of rosette-forming cells

Lymphocytes were isolated from human peripheral blood by carbonyl-iron treatment and Ficoll-Isopaque centrifugation. The lymphocytes were allowed to form rosettes with sheep erythrocytes, either uncoated (E) or coated with antibody and complement (EAC).In 32 experiments E rosettes were separated from free lymphocytes on a Ficoll density gradient. Thus, depleted (non-E) and enriched (E) fractions were obtained. It was found that in the original suspension 24 ± 7.2% of the lymphocytes formed rosettes with EAC and 56 ± 8% with E. In fraction non-E these values were 56 ± 11.4 and 22 ± 8.9%, respectively; in fraction E 8 ± 3.8 and 79 ± 8.8%. Moreover, the percentages of Ig-bearing cells among the fractions were found to follow closely those of CRL.In a series of lymphocyte culture experiments these fractions were compared with the original suspension and a control suspension (rosetted, not separated), as well as with a recombined population (non-E + E). It was found that fraction non-E showed an increased response to PHA and PWM, and an enhanced MLC stimulatory capacity, whereas fraction E was decreased in these respects. Moreover, fraction E displayed significantly reduced spontaneous DNA synthesis.It is concluded that the responses to PHA or PWM, as well as the capacity to stimulate allogeneic cells, are not solely dependent on the cells forming rosettes with E.     

Immunologic functions of isolated human lymphocyte subpopulations. IV. Stimulation of MLC and CML by human T cells.

Surface immunoglobulin positive and immunoglobulin negative human lymphocyte populations were obtained by immunoabsorbent column chromatography. Both cell populations were effective as stimulating and target cells in allogeneic MLC and CML reactions. The immunoglobulin negative population was further depleted of both EAC rosette forming cells and nylon wool adherent cells. The resulting highly purified T cell population was also able to stimulate allogeneic cells in MLC, and induce the generation of specifically cytotoxic killer cells in CML.