Subpopulations Of Blood Mononuclear Cells Research Articles

Stimulation and inhibition of human T cell subsets by wheat germ agglutinin.

Wheat germ agglutinin (WGA), a tetravalent lectin, has both stimulatory and inhibitory effects on human T lymphocytes. It has been suggested that these actions are related and that WGA selectively stimulates a suppressive subset of T cells. We studied the ability of WGA to stimulate and inhibit subpopulations of human peripheral blood mononuclear cells (PBMC) known to have helper or suppressor activity. Fresh human PBMC were depleted of either T4+ or T8+ cells by using antibody-mediated complement lysis. The resultant cell populations were stimulated with WGA, and the proliferative response was assessed by [3H]thymidine incorporation, IL 2 receptor expression, the ability to elaborate IL 2 in culture supernatants, and the susceptibility to inhibition by the monoclonal antibody anti-Tac. Similar experiments with cells from a WGA-responsive continuous T cell culture were also performed. WGA inhibited phytohemagglutinin (PHA)-induced proliferation of PBMC depleted of either T4+ or T8+ cells. WGA also inhibited PBMC that had been depleted of adherent cells and Ia+ cells and then induced to proliferate with a combination of TPA and PHA. Our findings indicate that WGA induces IL 2-dependent proliferation in a small proportion of both T4+ and T8+ lymphocytes. We also provide evidence that the inhibitory activity of WGA is not mediated by a T4+, T8+, or Ia+ cell, suggesting that WGA acts directly on the proliferating cell rather than selectively stimulating a suppressive subpopulation.

Autologous mixed lymphocyte reaction in patients with myasthenia gravis: correlation with disease activity.

Abstract Enriched subpopulations of peripheral blood mononuclear cells from patients with myasthenia gravis and from normal control individuals were tested for proliferative ability in the autologous mixed lymphocyte reactions. Myasthenia gravis patients were studied at various phases of therapy. Longitudinal patient populations were studied. Purified T cells from untreated patients with active myasthenia gravis demonstrated enhanced proliferative responses when co-cultured with nonmodified autologous non-T cells. Furthermore, treatment by thymectomy with or without prior plasmapheresis was associated with a consequent normalization of the autologous reaction. The enhanced autologous mixed lymphocyte reaction in the pre-treatment group of myasthenia gravis patients was significantly different from the post-treatment and normal control groups (Stimulation Index, p less than 0.01 and p less than 0.02, respectively). This difference in activity was also confirmed in a longitudinal study of 10 patients followed pre- and post-treatment (Stimulation Index, p less than 0.02). There was no difference between the responses of post-treatment patients and normal control individuals. There was no evidence of altered T cell responsiveness or non-T cell stimulation, as measured by the allogeneic mixed lymphocyte reaction or by mitogen-induced lymphocyte proliferation, between pretreatment and post-treatment patients as compared to controls. The responses were not affected by serum substitution experiments whereby myasthenic responder T cells were co-cultured with pooled normal human sera. Other parameters studied included clinical status, anti-acetylcholine receptor antibody levels, and thymic pathology. An intriguing connection between abnormalities in the thymic cell populations and the autoaggressive state in myasthenia may be reflected by the in vitro autologous mixed lymphocyte reaction.

Correlation of two in vitro tests with clinical response to immunosuppressive therapy in 54 patients with severe aplastic anemia

Two in vitro tests were applied to 54 consecutive patients with severe aplastic anemia who were treated in Seattle with antithymocyte globulin. In the first test, peripheral blood mononuclear cells were collected from each patient before antithymocyte globulin therapy and then treated with a panel of monoclonal antibodies and complement. The treated peripheral blood mononuclear cells were assayed for erythroid burst-forming units (BFU-E). This test was designed to determine whether removing various subpopulations of peripheral blood mononuclear cells would increase the number of detectable BFU-E. In the second test, peripheral blood was collected within 48 hr after completion of antithymocyte globulin therapy, and cells were immediately assayed for BFU-E without any further treatment. Data from both tests were analyzed to determine whether the in vitro results correlated with patient response to therapy. Binary logistic regression analyses indicate that a modest correlation (p = 0.04) exists between test 1 in vitro results and patient response to therapy. However, the strength of this association appears to decrease as the interval between diagnosis and treatment increases. In contrast, test 2 had a very significant correlation (p = 0.001) with response to therapy among patients diagnosed more than 1 mo prior to treatment, whereas such an association was not apparent among patients treated within 1 mo of diagnosis.

Correlation of Two In Vitro Tests With Clinical Response to Immunosuppressive Therapy in 54 Patients With Severe Aplastic Anemia

Two in vitro tests were applied to 54 consecutive patients with severe aplastic anemia who were treated in Seattle with antithymocyte globulin. In the first test, peripheral blood mononuclear cells were collected from each patient before antithymocyte globulin therapy and then treated with a panel of monoclonal antibodies and complement. The treated peripheral blood mononuclear cells were assayed for erythroid burst-forming units (BFU-E). This test was designed to determine whether removing various subpopulations of peripheral blood mononuclear cells would increase the number of detectable BFU-E. In the second test, peripheral blood was collected within 48 hr after completion of antithymocyte globulin therapy, and cells were immediately assayed for BFU-E without any further treatment. Data from both tests were analyzed to determine whether the in vitro results correlated with patient response to therapy. Binary logistic regression analyses indicate that a modest correlation (p = 0.04) exists between test 1 in vitro results and patient response to therapy. However, the strength of this association appears to decrease as the interval between diagnosis and treatment increases. In contrast, test 2 had a very significant correlation (p = 0.001) with response to therapy among patients diagnosed more than 1 mo prior to treatment, whereas such an association was not apparent among patients treated within 1 mo of diagnosis.

STUDY ON PERIPHERAL BLOOD MONONUCLEAR CELL SUBPOPULATIONS IN CHILDREN : Part 1.Peripheral Blood Mononuclear Cell Subpopulations in Healthy Neonates, Infants, Young Children and School Children

STUDY ON PERIPHERAL BLOOD MONONUCLEAR CELL SUBPOPULATIONS IN CHILDREN : Part 1.Peripheral Blood Mononuclear Cell Subpopulations in Healthy Neonates, Infants, Young Children and School Children

Spontaneous cytotoxicity by monocyte-enriched subpopulations of human peripheral blood mononuclear cells against human or mouse anchorage-dependent tumour cell lines. Contribution of NK-like cells.

Human peripheral blood mononuclear cells (PBMNC) were found to be cytotoxic for mouse or human anchorage-dependent target cell lines in a 48-72 h [125I]iododeoxyuridine (IUDR) release assay. Unfractionated, adherent or nonadherent cells had significant levels of cytotoxicity, as did cells fractionated according to size into 'lymphocytes' or 'monocytes' by elutriation. Intermediate size cells, not enriched for monocytes, had high levels of cytotoxicity. In all fractions tested, including adherent populations, some cells with the morphology of large granular cells were observed. Treatment of all fractions with interferon (IFLrA, a purified, recombinant alpha-IFN) boosted cytotoxicity against four target cells lines. Treatment with lymphokines containing putative 'macrophage-activating factor' (MAF) also enhanced cytotoxicity in fractions depleted of monocytes. Culture in fetal bovine serum enhanced cytotoxicity mainly in unfractionated and nonadherent PBMNC. These experiments indicated that NK-like cells can be appreciable contaminants in elutriator-purified monocyte-enriched or adherent cell populations and thereby contribute to observed cytotoxicity, particularly after pretreatment with IFN or other stimulatory factors.

The effect of a human plasma thymic factor on human peripheral blood mononuclear cell subpopulations

Human plasma thymic factor in vitro (human “facteur thymique sérique,” or FTS) (at concentrations between 0.25 and 0.25 × 10 5 pg/ml) significantly increased “avid” E-rosette formation by human peripheral blood mononuclear cells (PBMC). It also increased the percentage of OKT8 + cells but did not affect the percentages of OKT3 +, OKT4 +, OKT11 +, OKIa1 +, or OKM + cells. In the low-density immature PBMC, FTS increased both total and avid E-rosette formation, while increasing the percentages of OKT3 + and OKT8 + cells and decreasing the percentage of OKT4 + cells. Theophylline decreased E-rosette formation and the percentages of OKT3 + and OKT4 + among PBMC, and increased the percentages of OKT3 + and OKT4 + among the low-density cells. Human FTS may be capable of inducing maturation of immature PBMC into E-rosetteforming OKT3 +8 + cells, while increasing the percentage of mature cells which may be doubly labeled OKT4 +8 +. The function of such doubly labeled cells remains to be determined. Human FTS may act by increasing intracellular cAMP in immature cells, but probably has a different mode of action in mature cells.

The in vitro effect of phenytoin and carbamazepine on subpopulations of human blood mononuclear cells

Mononuclear cells from peripheral blood of 10 healthy blood donors were incubated with phenytoin and carbamazepine dissolved in 1% propyleneglycol. Phenytoin 20 mg/1 significantly reduced the percentage of active E rosette-forming T-lymphocytes; to mean 17.6% compared to mean 25.9% in control incubations ( P<0.05). Carbamazepine 10 mg/1 reduced this percentage to 21.0% (0.05< P<0.100). Phenytoin reduced the percentage of active T-lymphocytes in 9 of the 10 cell suspensions, while carbamazepine reduced the active T-lymphocytes in 8 of the 10 suspensions. The percentage of total E rosette-forming T-lymphocytes was similarly reduced by 20 mg/1 phenytoin and 10 mg/1 carbamazepine; from 65.3 to 59.5% and to 60.3%, respectively ( P<0.05, 0.05< P<0.10). The reduction of active and total T-lymphocytes was concentration-dependent for both drugs. Phenytoin and carbamazepine 5 mg/1 did not influence the percentage of T-lymphocytes. The decrease became apparent at a concentration of 10 mg/1 for both drugs, and grew steadily more marked with increasing drug concentrations up to 80 mg/1. However, the reduction in the percentage of T-lymphocytes was more pronounced for phenytoin than for carbamazepine at all drug concentrations. The percentage of cells with Fcγ-receptors and of those with receptors for the activated human complement component C3b, were not influenced by phenytoin and carbamazepine, nor were the cells with phagocytizing capacity and the non-phagocytizing cells with membrane-bound immunoglobulin (i.e. B-lymphocytes).

In vitro effect of ethanol on subpopulations of human blood mononuclear cells.

Mononuclear cells from 10 healthy blood donors were incubated with ethanol at 37 degrees C for 30 min. An ethanol concentration of 10.0 g/l reduced the percentage of active E rosette-forming cells (E RFC) from 25.0 +/- 12.1 to 16.2 +/- 8.7, and the percentage of total E RFC from 70.3 +/- 8.5 to 59.7 +/- 13.5 (p less than 0.01 for both). The percentage of cells with phagocytizing capacity was reduced from 11.7 +/- 5.4 to 7.0 +/- 5.0 by the ethanol treatment (p less than 0.01). Incubation with ethanol at a concentration of 1.0 g/l also significantly reduced the number of active and total E RFC and of phagocytizing cells. Ethanol at a concentration of 0.1 g/l did not influence these cell subpopulations. The number of EA RFC and EAC RFC were not influenced by ethanol.

Human erythroid burst-forming units. Growth in vitro is dependent on monocytes, but not T lymphocytes.

The roles of monocytes and T lymphocytes in the regulation of human peripheral blood erythroid burst-forming units (BFU-E) were studied in erythropoietin-containing plasma clot cultures of subpopulations of human blood mononuclear cells. BFU-E growth was decreased significantly after depletion of monocytes alone (mean 11% of expected, range 0 to 42% of expected) or depletion of both monocytes and T cells (mean 6.5% of expected, range 0.5 to 12% of expected) from mononuclear cells. T cell depletion did not impair BFU-E growth in vitro. Using 10(5) monocyte- and T lymphocyte-depleted mononuclear cells as target cells (less than 1% monocytes, less than 5% T cells), BFU-E growth was restored to 40% of expected by addition of 10(4) monocytes, and to 96% of expected by 10(5) monocytes alone. Addition of as many as 2 X 10(5) T cells but no monocytes resulted in stimulation to only 34% of expected BFU-E growth. Addition of 2 X 10(4) T cells, which alone did not affect BFU-E growth, could augment significantly the stimulatory effect of 5-20 X 10(3) monocytes on BFU-E growth. Thus, monocytes alone appear to be capable of stimulating BFU-E growth in vitro in the presence of erythropoietin. T cells also may make small quantities of BFU-E stimulators. However, it seems more likely that the most important role of T lymphocytes in BFU-E regulation in vitro is a result of interactions with monocytes and augmentation of monocyte production of stimulators of BFU-E growth.

Human T lymphcytes are enriched for transfer factor.

Using a canine local skin assay, we tested transfer factor prepared from three subpopulations of human peripheral blood mononuclear cells. T lymphocytes are enriched for transfer factor whereas adherent cells and nonadherent/nonrosetting cells are not. Thus, it appears as if transfer factor is a T cell lymphokine, the immunobiology of which is still unclear.

Activation of Human B Lymphocytes

Abstract The present study defines and characterizes multiple subpopulations of immunoregulatory human peripheral blood (PB) mononuclear cells of in vitro immunoglobulin (Ig) synthesis. The in vitro pokeweed mitogen-induced (PWM) anti-sheep red blood cell (anti-SRBC) plaque-forming cell (PFC) response and intracytoplasmic (ICP) Ig formation varied among normal subjects studied. On the basis of PFC responses, normal individuals can be divided into responders (&amp;gt;75 PFC/106 mononuclear cells) and nonresponders (&amp;lt;25 PFC/106 mononuclear cells). Furthermore, PB lymphocytes from PFC nonresponders also had decreased responses with regard to PWM-induced ICP Ig. Irradiated and nonirradiated unfractionated (total) T cells from responders both provided a net helper effect for PFC induction and ICP Ig production when co-cultured with allogeneic B cell-enriched suspensions. Within the total T cell population from responder individuals, T cells with an Fc receptor (R) for IgG (TG cells) suppressed PFC responses, whereas corresponding TG-depleted T cells obtained after overnight incubation did not suppress PFC responses. In addition, the total non-T cell fraction from responders suppressed B cell function in allogeneic and autologous co-cultures. However, this responder non-T cell-mediated suppression of Ig synthesis was radioresistant and was abrogated by removal of adherent cells. In contrast, nonirradiated total T cells from nonresponders did suppress PFC induction and ICP Ig production by allogeneic B cell-enriched suspensions. However, similar to responder individuals, irradiated total T cells from nonresponders not only did not suppress, but provided maximal T cell helper effect for PFC induction and ICP Ig production by allogeneic B cells. Furthermore, similar to responders, total non-T cell (E-rosette negative) fractions from nonresponders suppressed PFC responses in allogeneic co-cultures. However, this suppression was not abrogated by removal of adherent cells. The nonadherent suppressor cells in the E-rosette positive and E-rosette negative fraction from nonresponders bore an FcR for IgG. The primary difference between immunoregulatory cells found in responder and nonresponder subjects was the presence in nonresponders of E-rosette positive and E-rosette negative IgG FcR+ suppressor lymphocytes that did not require exposure to 7SEA to suppress. Thus, the functional capabilities of immunoregulatory cell populations differ greatly among normal individuals and relate primarily to immunoregulatory control of Ig synthesis at the T cell level. These studies illustrate the complexities of human immunoregulatory cell functions as expressed in in vitro cultures and illustrate that many of the in vitro immunoregulatory abnormalities associated with disease states can be demonstrated under certain conditions in mononuclear cell suspensions from normal subjects.

Activation of Human B Lymphocytes

Abstract Heterogeneous subpopulations of peripheral blood (PB) mononuclear cells that are capable of being activated by concanavalin A (Con A) to suppress the pokeweed mitogen (PWM)-induced anti-sheep red blood cell (SRBC) direct plaque-forming cell (PFC) response of human B cells have been described. Removal of adherent cells by passage over a Sephadex G-10 column or by plating of PB mononuclear cells on plastic surfaces did not affect the generation of Con A-activated suppressor cells (SC) (p &amp;lt; 0.01). Separation of PB cells into T cell-enriched (TCE) and T cell-depleted (TCD) suspensions by SRBC rosetting procedures demonstrated maximal Con A-activated suppression in each of these subpopulations (p &amp;lt; 0.001). Separation of the TCE fraction into T cells that bear Fc receptors for IgG (T.G) and those that do not (T.G-depleted) demonstrated submaximal suppression in each group alone. The T.G-depleted subpopulation suppressed to 50 ± 22% of control whereas the T.G fraction obtained by 7S EA rosetting did not suppress (128 ± 66% of control). However, T.G-enriched cells obtained, without activating the 7S Fc receptor, by depleting TCE fractions of T cells bearing an Fc receptor for IgM (T.M-depleted) did suppress maximally. Finally, separation of unfractionated PB mononuclear cells into those cells that possess complement (C) receptors (CR positive) and those that do not (CR negative), demonstrated that there was significant Con A-activated suppression in the CR-negative fraction of cells (p &amp;lt; 0.01) whereas the Con A-activated CR-positive cell did not suppress (p &amp;gt; 0.2). Thus, these studies demonstrate that cells that are capable of being activated by Con A to suppress the PWM-induced PFC response of normal human PB mononuclear cells are found in both the TCE and TCD fractions. In the TEC suspensions, Con A SC are found in both IgG Fc receptor positive and negative fractions, although IgG Fc receptor negative cells were less potent suppressors than IgG Fc receptor positive cells. In the TCD suspension, Con A SC are nonadherent, E-rosette-negative, Cr-negative cells.

Variations in the intracellular potentials of sub-populations of human peripheral blood mononuclear cells

The intracellular potential of human peripheral blood lymphocytes and monocytes was measured with a modified neurophysiological system and was found to vary between −20 mV and +20 mV. By depleting the total mononuclear fraction of monocytes and B-lymphocytes, and by using various separation procedures to derive monocyte, B-lymphocyte-, T-lymphocyte-, and null cell-enriched populations, it was possible to show that monocytes and the majority of the B-lymphocytes had positive intracellular potentials, whereas T-lymphocytes and null cells had negative intracellular potentials. Various factors, which include in vitro culturing, PHA and Con A stimulation, and possibly maturity, significantly affect the intracellular potential.

Purification and Characterization of Granulocytic Progenitor Cells (CFU-C) From Human Peripheral Blood Using Immunologic Surface Markers

The concentration of committed granulocytic progenitor cells (CFU-C) in functionally unique subpopulations of human peripheral blood mononuclear cells has been determined by the in vitro methyl-cellulose assay. Using immunoabsorbent column chromatography and rosette-depletion techniques, we have demonstrated that CFU-C, although not present in either purified T or B lymphocyte populations, are highly concentrated in the "null" cell population, which lacks sheep erythrocyte receptors and surface immunoglobulin. Further fractionation of this null subset has demonstrated that CFU-C do not bear complement receptors, but require the presence of peripheral blood mononuclear cell feeder layers for maximum proliferation.

Purification and characterization of granulocytic progenitor cells (CFU- C) from human peripheral blood using immunologic surface markers

The concentration of committed granulocytic progenitor cells (CFU-C) in functionally unique subpopulations of human peripheral blood mononuclear cells has been determined by the in vitro methyl-cellulose assay. Using immunoabsorbent column chromatography and rosette-depletion techniques, we have demonstrated that CFU-C, although not present in either purified T or B lymphocyte populations, are highly concentrated in the “null” cell population, which lacks sheep erythrocyte receptors and surface immunoglobulin. Further fractionation of this null subset has demonstrated that CFU-C do not bear complement receptors, but require the presence of peripheral blood mononuclear cell feeder layers for maximum proliferation.

Spontaneous Cell-Mediated Cytotoxicity by Human Peripheral Blood Lymphocytes in Vitro

Abstract With isolated subpopulations of human peripheral blood mononuclear cells, spontaneous cell-mediated cytotoxicity (SCMC) and antibody-dependent cellular cytotoxicity (ADCC) were demonstrated to be functions of a population of surface immunoglobulin (sIg)-negative, Fc receptor-positive lymphocytes. Organ distribution studies revealed that SCMC and ADCC effector cell activity occurred in parallel, being present in spleen and absent in tonsils. Treatment of effector cells with trypsin significantly inhibited SCMC but not ADCC and this inhibition was not reversed by short-term incubation in autologous plasma. These results demonstrate that SCMC and ADCC involve different cytotoxicity mechanisms and are consistent with two subsets of effector cells with separate functional capabilities or a single effector cell mediating both forms of cytotoxicity through separate mechanisms.