Human Peripheral Blood Lymphocyte Subpopulations Research Articles

Prospects and Methods for Studying the Proliferative Capacity of the Human Peripheral Blood Lymphocyte Subpopulations in Radiation Medicine

Prospects and Methods for Studying the Proliferative Capacity of the Human Peripheral Blood Lymphocyte Subpopulations in Radiation Medicine

Reference intervals for human peripheral blood lymphocyte subpopulations from 'healthy' young and aged subjects.

Some lymphocyte subpopulations change during aging but age-related reference limits from a healthy reference population are still lacking. In this study, we compiled 90% reference intervals for commonly determined lymphocyte subpopulations in 'healthy' (Senieur-compatible) young (20-32 years) and elderly (65-74 years) subjects. The most striking age-related changes included increases in HLA-DR+ T lymphocytes, and the shift in the expression of CD45 isoforms from the CD45RA+CD45RO-to the CD45RA-CD45RO+ subset. Both age-related alterations occurred in the CD4+ as well as in the CD8+ subpopulations and most of them were present in the relative and absolute number of lymphocyte subsets. We compare our data with those from previous investigations on lymphocyte subpopulations from the elderly and comment on useful presentation of reference values.

Lack of direct immunosuppressive effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on human peripheral blood lymphocyte subsets in vitro.

A wide variety of immunosuppressive effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in experimental animals has been documented. In contrast, the impact of dioxin on the human immune system remains controversial, although adverse health effects have been reported in humans after occupational or accidental exposure to dioxin. Recently, Neubert et al. (1991) found that a dose-dependent decrease of peripheral blood lymphocyte (PBL) subpopulations in humans and non-human primates, including helper-inducer/memory cells (CD4+CD29+) and B cells (CD20+) occurred in pokeweed mitogen (PWM) stimulated cultures at concentrations as low as 10(-12)-10(-14) M TCDD. Therefore, the direct effects of dioxin on human PBL subpopulations have been studied, in order to determine their usefulness as sensitive biomarkers for human dioxin exposure. Lymphocyte cultures from healthy individuals were treated with 10(-7) M-10(-14) M TCDD in the absence and presence of stimulation with pokeweed mitogen (PWM) or anti-CD3 monoclonal antibody (moAb; OKT3) for 3 days. Cytochrome P450 (CYP1A1) enzyme induction, one of the best studied direct biological effects of TCDD on numerous cell types, was assayed in parallel by ethoxyresorufin-O-deethylase (EROD) activity. Percentages of the different lymphocytes subsets, including CD2 (T cells); CD4; CD45 RA (suppressor-inducer/virgin T cells); CD4 CD29; CD8; CD19 (B cells) as well as interleukin 2 (IL-2) receptor (CD25) and class II antigen (HLA-DR) expression, were analyzed by flow cytometry. DNA synthesis was determined by 3H-thymidine uptake after 3 days of culture.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Expression of perforin and cytolytic potential of human peripheral blood lymphocyte subpopulations.

To verify the physiological role of the pore-forming protein perforin in vivo, its expression in subpopulations of human peripheral blood lymphocytes was examined by immunocytochemical staining and their cytolytic potentials compared. In addition to NK cells and gamma delta T cells, which uniformly expressed abundant perforin in their cytoplasmic granules, only a small subpopulation of CD8+ alpha beta T cells contained perforin, namely the CD11b+ subset. However, in vitro activation with an anti-CD3 antibody and IL-2 induced perforin expression in approximately 50% of the CD8+CD11b- T cells and also in a small subset of CD4+ T cells. A distribution of perforin in CD8+ and CD4+ T cells, similar to in vitro activated T cells, was observed in fresh peripheral blood lymphocytes from infectious mononucleosis patients. In all instances, the expression of perforin correlated with the cytolytic potential of these subpopulations. The results strongly suggest that perforin plays a role in the manifestation of cytotoxic activity in vivo.

The stimulation of human peripheral blood lymphocytes by solubilized dental plaque: macrophage and T-cell dependence.

This study compared the ability of human peripheral blood lymphocyte subpopulations from individuals with moderate periodontitis to proliferate in response to stimulation with solubilized dental plaque, phytohemagglutinin (PHA), pokeweed mitogen (PWM) and purified protein derivative (PPD). Unseparated mononuclear lymphocytes and recombined purified T and B lymphocytes (4/1) responded significantly to all of the stimulants. DNA synthesis in response to solubilized dental plaque was maximal after 6 to 7 days of culture; the optimum dose was usually 10 to 20 micrograms/ml. T lymphocyte subpopulations purified by rosetting with sheep red blood cells and density gradient centrifugation responded well to PHA and PWM, but were unresponsive to solubilized dental plaque and PPD unless supplemented with 2% autologous macrophages, demonstrating that T cell responses to solubilized dental plaque and to PPD are macrophage-dependent. T cell-depleted enriched B lymphocyte subpopulations were poorly responsive to all of the tested stimulants; however, the responses of these cells to solubilized dental plaque and to the known T cell-dependent B cell mitogens PWM and PPD were increased significantly by the presence of 10% mitomycin C-treated T cells, demonstrating that B cell proliferation to solubilized dental plaque is T cell-dependent. Thus, cellular interactions between macrophages, T lymphocytes and B lymphocytes are required to obtain optimal proliferative lymphocyte responses to solubilized dental plaque. Since both T and B lymphocytes respond to dental plaque stimulants, they both have the potential to mediate periodontal inflammation and tissue destruction whenever dental plaque stimulants gain entrance into the periodontal tissues.

Expression of the Chediak-Higashi lysosomal abnormality in human peripheral blood lymphocyte subpopulations

Fusion of lysosomes to form a giant cytoplasmic inclusion is a major abnormality expressed by multiple hematopoietic and non-hematopoietic cell types in Chediak-Higashi (C-H) patients. In this study, the extent of involvement of lymphoid cell subpopulations was defined. Purified populations of B cells, natural killer (NK) cells, and helper T cells were obtained from two C-H patients and normal controls by immunofluorescence staining of their blood mononuclear cells with the monoclonal antibodies HB-2, Leu-7, or Leu-3 followed by fluorescence- activated cell sorting. Cytochemical and ultrastructural analyses as well as functional assays were performed to determine whether or not the C-H lysosomal abnormality was expressed in the different lymphocyte subpopulations. B cells expressed the C-H defect following activation and differentiation. All of the Leu-7+ cells and a significant proportion of the Leu-3+ cells displayed the C-H abnormality. These Leu- 3+ cells share the NK lineage characteristics of granular lymphocyte morphology and the capacity to bind to NK cell targets. In contrast, the C-H abnormality was not observed in non-NK target-binding cells with T helper phenotype, in which clusters of lysosomes formed a normal Gall body. Moreover, T cell functions were unimpaired in C-H patients. These observations raise the issue of the lineal relationship between granular and nongranular lymphocytes typed as T cells on the basis of cell surface antigen markers.

Expression of the Chediak-Higashi Lysosomal Abnormality in Human Peripheral Blood Lymphocyte Subpopulations

Fusion of lysosomes to form a giant cytoplasmic inclusion is a major abnormality expressed by multiple hematopoietic and non-hematopoietic cell types in Chediak-Higashi (C-H) patients. In this study, the extent of involvement of lymphoid cell subpopulations was defined. Purified populations of B cells, natural killer (NK) cells, and helper T cells were obtained from two C-H patients and normal controls by immunofluorescence staining of their blood mononuclear cells with the monoclonal antibodies HB-2, Leu-7, or Leu-3 followed by fluorescence-activated cell sorting. Cytochemical and ultrastructural analyses as well as functional assays were performed to determine whether or not the C-H lysosomal abnormality was expressed in the different lymphocyte subpopulations. B cells expressed the C-H defect following activation and differentiation. All of the Leu-7+ cells and a significant proportion of the Leu-3+ cells displayed the C-H abnormality. These Leu-3+ cells share the NK lineage characteristics of granular lymphocyte morphology and the capacity to bind to NK cell targets. In contrast, the C-H abnormality was not observed in non-NK target-binding cells with T helper phenotype, in which clusters of lysosomes formed a normal Gall body. Moreover, T cell functions were unimpaired in C-H patients. These observations raise the issue of the lineal relationship between granular and nongranular lymphocytes typed as T cells on the basis of cell surface antigen markers.

Catechol- O-methyltransferase activity and aminergic binding sites distribution in human peripheral blood lymphocyte subpopulations

We have studied the binding of [ 3H]dihydroalprenolol, a β-adrenergic ligand, and [ 3H]spiroperidol, a dopaminergic ligand, to human peripheral blood lymphocyte subpopulations. The catechol- O-methyltransferase activity on these different cells was also determined. On intact living cells, an unequal distribution of both β-adrenergic and dopaminergic binding sites was found between B- and T-lymphocyte subpopulations. In each binding, T cells exhibited a lower number of binding sites than B cells. On particulate preparation, catechol- O-methyltransferase activity showed a similar distribution, but binding data with such material were quite different. Although it was not possible to detect dopaminergic binding sites on particulate preparation, T and B lymphocytes contained equivalent numbers of β-adrenergic binding sites. Different explanations for these results are proposed.

Image processing in pathology: XI. Light microscopic discrimination of human peripheral blood lymphocyte subpopulations

Non-labelled lymphocytes in peripheral blood smears and in smears of purified lymphocyte suspensions were differentiated in T- and non-T-cells (controlled by E-rosetting assay and surface immunoglobulin detection) by automatic image processing at the light microscopic level. By various classification methods it was found that these lymphocyte subpopulations differ in several visual and subvisual morphologic features. The morphologic differences between subpopulations were superimposed by individual deviations as well as by influences from the preparation. Single features were strongly affected by exogenic factors. Most of the different features are comparable with fine structural differences, as previously reported.

Cooperative Effects in Mitogen- and Antigen-Induced Responses of Human Peripheral Blood Lymphocyte Subpopulations

Human peripheral blood lymphocytes were separated into T cell-enriched and T cell-depleted fractions by E rosette sedimentation. These two fractions, as well as the unseparated lymphocyte suspension, were tested for their responsiveness to the mitogens phytohemagglutinin (PHA), concanavalin A (ConA) and pokeweed mitogen (PWM) and to the antigens PPD (purified protein derivative of tuberculin) and tetanus toxoid. The response to PHA, ConA and the antigens was found to be confined to the purified T cell fraction; PWM could stimulate both purified T and non-T cells. However, the T cell response to ConA, PPD and tetanus toxoid was always decreased by 50-70%, when compared to the unseparated lymphocytes. Addition of monocytes could restore the T cell response. In the response to PHA and tetanus toxoid, the (primarily unresponsive) non-T cell fraction could be recruited into proliferation by gamma-irradiated T cells. Moreover, in the response to tetanus toxoid, lymphocytes (T as well as non-T) from a nonimmune individual could be recruited into proliferation by gamma-irradiated immune T cells.

Human B-cell mitogenic responsiveness to lectins: The requirement for T cells

The mitogenic potential of normal human peripheral blood lymphocyte subpopulations in response to the plant lectins phytohemagglutin, erythroaggluting phytohemagglutinin, leukoagglutinating phytohemagglutinin, and pokeweed mitogen has been examined. Doubly purified B-, Null, and T-cell fractions were used by themselves and in conjunction with small percentages (1 to 10%) of other subpopulations. Purified human B and Null cells were found to be nonreactive. However, when incubated with as little as 1% T cells, significant mitogenic responsiveness by B and Null cells was observed. This helper effect was apparent with both normal and irradiated T cells. It would thus appear that mitogenic responsiveness of unseparated mononuclear cells is predominantly a function of T cells either as responding cells or as necessary helper cells.

Delineation and Quantitation of Human Peripheral Blood Lymphocyte Subpopulations by Electrophoretic Mobility and Role of Surface Charge in Cell to Cell Interaction

A modified procedure is proposed for determination of electrophoretic mobility (EPM) of lymphocytes which enables readings on 200 cells from a sample size of 5 x 106 lymphocytes. In normal human peripheral blood, B lymphocytes (bearing surface immunoglobulin and receptor for activated C3) were found to carry the lowest charge (0.94 +/- 0.05 mu/sec/V/cm). Their number on the EPM basis was 16.9 +/- 4.2%. Two subpopulations of T lymphocytes, one carrying high charge (TH) with a mean EPM of 1.26 +/- 0.04 mu/sec/V/cm and the other carrying lower charge (TL) with a mean EPM of 1.11 +/- 0.03 mu/sec/V/cm were discernible. The former subpopulation of T cells formed spontaneous rosettes with SRBC in 10 min and the latter represented the remaining T lymphocytes which formed rosettes with 24 hr of incubation with SRBC. The number of early rosette-forming cells increases from 69 to 89% when T cells were incubated with neuraminidase-treated SRBC. These observations suggest that complementary electrostatic charge on T cells and SRBC are among the forces facilitating formation of rosettes between these cells.