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Biomedical subjects

L F Thompson

Publications and source records attributed to L F Thompson.

At least 55 records · Page 3Linked to original sources
PubMed1990

Human T cell activation. Synergy between CD73 (ecto-5'-nucleotidase) and signals delivered through CD3 and CD2 molecules.

Interaction of the glycosyl phosphatidylinositol-linked differentiation Ag CD73 (ecto-5'-nucleotidase) with the CD73-specific mAb 1E9 generates agonistic signals that strongly synergize with T cell activation induced by CD3 and CD2 mAb. This synergy is observed only when 1E9 is immobilized on plastic and occurs in the absence of accessory cells or exogenous lymphokines. 1E9 induces a rapid (though transient) increase in [Ca2+]i in a minor proportion (20 to 30%) of unfractionated T lymphocytes (presumably CD73+ cells). However, this [Ca2+]i mobilization is not sufficient to fully activate CD73+ T cells, as shown by the requirement of additional signals such as CD3 or CD2 stimulation to initiate T cell proliferation. These signals cannot be substituted by the exogenous lymphokines, rIL-1, rIL-2, or rIL-4, or PMA (when T cells are rigorously depleted of monocytes). These data indicate that CD73 may behave as an accessory molecule regulating interactions between T cells and antigens or APC. A comparison was carried out with mAb 9.3 to the differentiation Ag CD28, another agonistic molecule with activating properties similar to CD73. Despite their lower percentage, the ability of CD73+ T cells to amplify the proliferation induced by CD3 or CD2 mAb was equivalent or even greater than that of CD28+ T cells. Once activated, CD73+ cells may recruit the remaining (CD73-) cells primed by CD3 or CD2 stimulation. Based on these data, we suggest that CD73+ T lymphocytes may be a specialized subset to amplify immune responses originated by the CD3 and CD2 activation pathways. Finally, the functional association between CD73 and integral membrane molecules like CD3 and CD2 suggests that GPI-anchored molecules may play a role in transmembrane signaling mediated by conventional second messenger systems.

5'-Nucleotidase↗
PubMed1989

Antibodies to 5'-nucleotidase (CD73), a glycosyl-phosphatidylinositol-anchored protein, cause human peripheral blood T cells to proliferate.

Human peripheral blood T cells were stimulated to proliferate when cultured with submitogenic doses of PMA and goat antibodies to 5'-nucleotidase (5'-NT). The degree of proliferation, as measured by [3H]TdR incorporation on day 3, was similar to that achieved by stimulation with PHA. Anti-5'-NT antibodies had no effect on PHA-induced proliferation. Maximal stimulation was achieved with 0.6 to 1.0 ng/ml of PMA and 125 micrograms/ml of IgG isolated from a goat anti-5'-NT antiserum. Both intact IgG and F(ab')2 fragments were stimulatory. IL-2R expression and IL-2 secretion were also induced by anti-5'-NT antibodies and PMA. Anti-5'-NT-induced proliferation was inhibited greater than 95% by a murine anti-IL-2 receptor mAb and required less than 0.3% monocytes. Similar results have been obtained with a murine mAb specific for 5'-NT. As expected, anti-5'-NT antibodies and PMA did not induce the proliferation of ecto-5'-NT-T cells isolated by cell sorting. Pretreatment of total T cells with phosphatidylinositol-specific phospholipase C removed an average of 89% of the 5'-NT activity from the cell surface and also inhibited by 83% the ability of the cells to proliferate in response to anti-5'-NT antibodies and PMA. Thus, the activation signal provided by anti-5'-NT antibodies is apparently transduced, in large part, by a form of the enzyme that is attached to the membrane via glycosyl-phosphatidylinositol linkage. These data suggest that 5'-NT may play a role in lymphocyte activation as has been proposed for other glycosyl-phosphatidylinositol-anchored lymphocyte surface proteins.

5'-Nucleotidase↗
PubMed1989

Functional characterization of ecto-5'-nucleotidase-positive and -negative human T lymphocytes.

Functional studies were performed on human peripheral blood T lymphocytes stained with goat anti-5'-nucleotidase antibodies and separated into ecto-5'-nucleotidase (ecto-5'-NT)-positive and -negative populations using the FACSTAR fluorescence-activated cell sorter. On the average, ecto-5'-NT+ T cells contained 34 +/- 13% CD4+ and 55 +/- 15% CD8+ cells, whereas ecto-5'-NT-T cells contained 65 +/- 12% CD4+ and 23 +/- 8% CD8+ cells. Staining with anti-5'-NT antibodies did not significantly alter the ability of unseparated T cells to proliferate in response to PHA or PMA, or in a MLR. However, prior incubation with anti-5'-NT antibodies did inhibit the ability of irradiated T cells to provide help for PWM-stimulated Ig synthesis by as much as 55%. In five separate experiments, ecto-5'-NT-T cells demonstrated an equal or better ability to incorporate [3H]TdR after PHA stimulation or in a MLR, as compared with ecto-5'-NT+ T cells. Similarly, ecto-5'-NT- T cells were not diminished in their ability to provide help for autologous B cells in a PWM-driven system. Clearly, the inability of ecto-5'-NT- T cells from patients with a variety of immunodeficiency diseases to function in these assays cannot be explained solely by their lack of ecto-5'-NT activity. In contrast, ecto-5'-NT-positive and -negative T cells showed markedly different dose-response curves for proliferation in response to PMA. Ecto-5'-NT+ T cells responded to lower doses of PMA (1.0 ng/ml) than did ecto-5'-NT- T cells and showed a two- to eight-fold greater rate of [3H]TdR incorporation at 3 to 10 ng of PMA per ml. Ecto-5'-NT+ T cells may have a protein kinase C that is more accessible or more easily activated or may utilize an alternate pathway of activation when stimulated with low concentrations of PMA.

5'-Nucleotidase↗
PubMed1988

Synthesis of immunoglobulin G by pokeweed mitogen- or Epstein-Barr virus-stimulated human B cells in vitro is restricted to the ecto-5'-nucleotidase positive subset.

Ecto-5'-nucleotidase (ecto-5'-NT) is believed to be a maturation marker for human B lymphocytes because its expression increases during normal development and is reduced in many patients with B cell immunodeficiencies. To determine whether this enzyme defines functional subsets of B lymphocytes, human peripheral blood B cells, separated into ecto-5'-NT positive and negative populations by using goat anti-5'-NT antibodies and the fluorescence-activated cell sorter, were compared for their ability to secrete polyclonal immunoglobulin. Both populations synthesized equivalent quantities of IgM in response to a T cell-dependent (PWM) or T cell-independent (EBV) stimulator of polyclonal immunoglobulin biosynthesis. However, ecto-5'-NT+ B lymphocytes synthesized 8- to 26-fold more IgG per cell than ecto-5'-NT- B cells. These data provide the first direct evidence that ecto-5'-NT is a marker for the functional maturation of human B cells and support the hypothesis that ecto-5'-NT deficiency in patients with hypogammaglobulinemia results from a block in B lymphocyte maturation.

5'-Nucleotidase↗
PubMed1987

Distribution of ecto-5'-nucleotidase on subsets of human T and B lymphocytes as detected by indirect immunofluorescence using goat antibodies.

Human T and B lymphocyte subsets were characterized for ecto-5'-nucleotidase (ecto-5'-NT) expression by two-color immunofluorescence by using polyclonal goat antibodies to 5'-NT and murine monoclonal antibodies to T and B cell subsets. Anti-5'-NT antibodies were prepared by immunizing a goat with purified human placental 5'-NT. Lymphocyte surface 5'-NT was detected with F(ab')2 fragments of immune goat IgG followed by biotinylated F(ab')2 rabbit anti-goat IgG and fluorescein isothiocyanate-avidin. Lymphocyte cell surface antigens were detected with phycoerythrin (PE)-conjugated anti-CD3, anti-CD4, anti-CD8, anti-CD16, and anti-CD19. HB-4, an antigen present on a major subset of human peripheral blood B cells, was detected with murine monoclonal anti-HB-4 and PE-anti-mouse-kappa. Analysis showed that ecto-5'-NT was expressed on 32 +/- 7% of CD3+, 19 +/- 6% of CD4+, and 50 +/- 21% of CD8+ T cells, but not on CD16+ lymphocytes. Ecto-5'-NT was also expressed on 81 +/- 8% of adult peripheral blood B cells as defined by PE-anti-CD19; HB-4 was expressed on 84 +/- 7% of CD19+ cells. The two populations of B cells were not identical, however, because HB-4 was co-expressed on only 79 +/- 18% of ecto-5'-NT+ B cells. Two-color immunofluorescent staining of T cells from a patient with congenital agammaglobulinemia and low T cell ecto-5'-NT activity revealed reduced percentages of ecto-5'-NT+ cells in his CD3+, CD4+, and CD8+ populations. Thus, reduced ecto-5'-NT activity by enzyme assay was paralleled by reduced numbers of 5'-NT molecules on the cell surface. Two-color immunofluorescent staining of B cells from a patient with hypogammaglobulinemia and low B cell ecto-5'-NT activity also revealed markedly reduced expression of 5'-NT. HB-4 expression was normal, however, suggesting that the patient's B cells were blocked in maturation subsequent to the acquisition of HB-4 but prior to that of ecto-5'-NT. These results demonstrate that anti-5'-NT antibodies will be valuable tools for analyzing ecto-5'-NT expression and lymphocyte maturation in patients with immuno-deficiency diseases.

5'-Nucleotidase↗
PubMed1987

Purification of 5'-nucleotidase from human placenta after release from plasma membranes by phosphatidylinositol-specific phospholipase C.

5'-Nucleotidase was purified greater than 1000-fold from human placenta by treatment of plasma membranes with S. aureus phosphatidylinositol-specific phospholipase C and affinity chromatography on Con A Sepharose and AMP-Sepharose. The resulting enzyme had a specific activity of greater than 5000 mumol/hr/mg protein and a subunit molecular weight of 73,000. Goat antibodies against 5'-nucleotidase inhibited enzyme activity and detected 5'-nucleotidase after Western blotting. These antibodies also recognized a soluble form of 5'-nucleotidase and residual membrane-bound 5'-nucleotidase which could not be released by phosphatidylinositol-specific phospholipase C treatment, suggesting that the three forms of the enzyme are structurally related. The soluble 5'-nucleotidase may be derived from the membrane-bound form by the action of an endogenous phospholipase C. The structural basis for the inability of some of the membrane-bound 5'-nucleotidase to be released by phosphatidylinositol-specific phospholipase C is unknown.

5'-Nucleotidase↗
PubMed1986

Ecto-5'-nucleotidase expression during human B cell development. An explanation for the heterogeneity in B lymphocyte ecto-5'-nucleotidase activity in patients with hypogammaglobulinemia.

Ecto-5'-nucleotidase (ecto-5'-NT) activity was measured in human B cells at different stages of development. Ecto-5'-NT activity of B cell preparations from fetal spleen and cord blood was 5.08 and 5.59 +/- 2.8 nmol/hr/10(6) cells, respectively; that of B cell preparations from adult peripheral blood, spleen, or lymph node was fivefold to sixfold higher (27.9 +/- 12, 29.2 and 33.8 nmol/hr/10(6) cells, respectively). The increased enzyme activity in B cell preparations from adult peripheral blood as compared with cord blood paralleled increased percentages of 5'-NT+ cells (69 +/- 12% vs 32 +/- 17%) and an average of twice as much enzyme activity per positive cell. Small, resting B cells that cannot synthesize Ig in vitro in response to pokeweed mitogen (PWM) were isolated from adult peripheral blood by mouse erythrocyte rosetting. Total ecto-5'-NT activity and the percentage of 5'-NT+ cells were equivalent in total B cells and the mouse erythrocyte rosette-positive subpopulation. Thus, ecto-5'-NT activity is acquired before B cells gain the ability to differentiate into Ig-secreting plasma cells in response to PWM. Ecto-5'-NT activity was also measured in B cell preparations from eight patients with common variable immunodeficiency. Six had reduced ecto-5'-NT activity (2.83 to 15.4 nmol/hr/10(6) cells), and two had normal activity (34.7 and 58.2 nmol/hr/10(6) cells). B cells from all six patients with low ecto-5'-NT activity failed to synthesize Ig when cultured with PWM and normal irradiated T cells. Of the two patients with normal B cell ecto-5'-NT activity, one also had B cells unresponsive to PWM, but B cells from the other patient appeared to more normal, in that they synthesized IgM and IgG when cultured with PWM plus irradiated allogeneic T cells. Thus, measurement of B cell ecto-5'-NT activity allows the subclassification of patients who have a common inability to synthesize immunoglobulin in vitro response to PWM. B cells with low ecto-5'-NT activity are presumably blocked at an earlier stage in development than B cells with normal ecto-5'-NT activity. Evaluation of ecto-5'-NT activity along with the expression of other B cell surface antigens should aid in the definition of discrete stages of B cell development.

5'-Nucleotidase↗
PubMed1985

IgE Fc receptor positive T, B and NK cells in patients with the hyper-IgE syndrome.

Patients with the hyper-IgE syndrome have greatly elevated percentages of IgE Fc receptor (Fc epsilon R)-positive B cells, but they have less than 0.1% Fc epsilon R+ T cells (T epsilon cells) and few, if any, Fc epsilon R+ natural killer cells. They also have markedly decreased numbers of IgG receptor positive (Fc gamma R+) T cells (T gamma cells). Patients with the hyper-IgE syndrome resemble in this respect patients with severe atopic dermatitis. Since a portion of T epsilon and T gamma cells of mildly atopic patients react with monoclonal antibody OKT8, they may have a suppressor function. However, whether the low number of T epsilon cells is responsible for the high IgE serum level in hyper-IgE syndrome and atopic dermatitis patients remains to be demonstrated. Attempts to obtain a reliable assay for human IgE synthesis in vitro to investigate the function of Fc epsilon R-positive lymphocytes proved to be difficult. Even isolated B cells from atopic donors seldom produced more than twice the quantity of IgE released from cells incubated in the presence of the protein synthesis inhibitor cycloheximide.

B-Lymphocytes↗
PubMed1985

Inosine 5'-monophosphate vs inosine and hypoxanthine as substrates for purine salvage in human lymphoid cells.

The ability of inosine 5'-monophosphate vs inosine or hypoxanthine to supply the total purine requirements of mitogen-stimulated human T cells or rapidly dividing human B lymphoblastoid cells was evaluated. Mitogen-stimulated human peripheral blood T cells were treated with aminopterin to inhibit purine synthesis de novo and make the cells dependent upon an exogenous purine source. Thymidine was added as a source of pyrimidines. Under these conditions, 25 microM inosine 5'-monophosphate, inosine, and hypoxanthine showed comparable abilities to support [3H]thymidine incorporation into DNA at rates equal to that of untreated control cultures. In parallel experiments with the rapidly dividing human B lymphoblastoid cell line, WI-L2, treatment with aminopterin (plus thymidine) inhibited the growth rate by greater than 95%. The normal growth rate was restored by the addition of 30 microM inosine 5'-monophosphate, inosine, or hypoxanthine to the medium. However, in similar experiments with cell line No. 1254, a derivative of WI-L2 which lacks detectable ecto-5'-nucleotidase activity, only inosine and hypoxanthine (plus thymidine), but not inosine 5'-monophosphate (and thymidine) were able to restore the growth inhibition due to aminopterin. These results show that the catalytic activity of ecto-5'-nucleotidase is sufficient to meet the total purine requirements of mitogen-stimulated human T cells or rapidly dividing human B lymphoblastoid cells and suggest that this enzyme may have functional significance when rates of purine synthesis de novo are limited and/or an extracellular source of purine nucleotides is available.

5'-Nucleotidase↗
PubMed1985

Ecto-5'-nucleotidase can provide the total purine requirements of mitogen-stimulated human T cells and rapidly dividing human B lymphoblastoid cells.

The ability of mitogen-stimulated human T cells or rapidly dividing human B lymphoblastoid cells to drive their total purine requirements from inosine 5'-monophosphate, inosine, or hypoxanthine was compared. Inosine 5'-monophosphate first must be converted to inosine by the action of the enzyme ecto-5'-nucleotidase before it can be transported into the cell; inosine and hypoxanthine, however, can be transported directly. Mitogen-stimulated human peripheral blood T cells were treated with aminopterin to inhibit purine synthesis de novo and to make the cells dependent on an exogenous purine source. Thymidine was added as a source of pyrimidines. Under these conditions, 30 microM inosine 5'-monophosphate, inosine, and hypoxanthine showed comparable abilities to support [3H]thymidine incorporation into DNA or [3H]leucine incorporation into protein at rates equal to that of untreated control cultures. Similar results were found when azaserine was used to inhibit purine synthesis de novo, and thus DNA synthesis. In parallel experiments with the rapidly dividing human B lymphoblastoid cell line WI-L2, treatment with aminopterin (plus thymidine) inhibited the growth rate by greater than 95%. The normal growth rate was restored by the addition of 30 microM inosine 5'-monophosphate, inosine, or hypoxanthine to the medium. However, in similar experiments with cell line 1254, a derivative of WI-L2 which lacks detectable ecto-5'-nucleotidase activity, inosine and hypoxanthine (plus thymidine), but not inosine 5'-monophosphate (and thymidine) were able to restore the growth inhibition due to aminopterin. These results show that the catalytic activity of ecto-5'-nucleotidase is sufficient to meet the total purine requirements of mitogen-stimulated human T cells or rapidly dividing human B lymphoblastoid cells, and suggest that this enzyme may be important for purine salvage when rates of purine synthesis de novo are limited and/or an extracellular source of purine nucleotides is available.

5'-Nucleotidase↗
PubMed1985

IgE Fc receptor positive T and B lymphocytes in patients with the hyper IgE syndrome.

The percentages of peripheral blood lymphocytes (PBL), bearing Fc receptors for IgE (Fc epsilon R) and IgG (Fc gamma R) were determined in four patients with the hyper IgE syndrome by a rosette assay employing IgE and IgG coated fixed ox erythrocytes. The patients had 8 +/- 3% Fc epsilon R+ and 13 +/- 8% Fc gamma R+ PBL, compared to 1.2 +/- 1% Fc epsilon R+ and 17 +/- 4% Fc gamma R+ PBL for control donors. T cells were isolated by rosetting with neuraminidase treated sheep erythrocytes (EN). Indirect immunofluorescence with Lyt 3 monoclonal antibody (MoAb) to the sheep erythrocyte receptor, followed by rosetting for Fc epsilon R and Fc gamma R showed that the patients' T cells contained less than 0.1% Fc epsilon R+ and 1.4 +/- 0.2% Fc gamma R+ cells; T cells from the control subjects contained less than 0.1% Fc epsilon R+ and 11 +/- 4% Fc gamma R+ cells. The non-T (EN rosette depleted) cells of the patients included 56 +/- 18% sIgM+/sIgD+, 45 +/- 9% Fc epsilon R+ and 35 +/- 27% Fc gamma R+ cells. Indirect immunofluorescence with MoAb to IgM, IgD, and NK cells (antibody B73.1) followed by rosetting for Fc epsilon R and Fc gamma R, indicated that 92 +/- 2% of the Fc epsilon R+ cells and 9 +/- 7% of the Fc gamma R+ cells were B cells (mu+/delta+), while 3 +/- 4% of the Fc epsilon R+ and 30 +/- 23% of the Fc gamma R+ cells were NK cells (B73.1+). Thus, most of the Fc epsilon R+ non-T cells were B cells, and only a small fraction appeared to be NK cells. On the other hand, Fc gamma R+ B cells were outnumbered by Fc gamma R+ NK cells (B73.1+) by three to one. The data indicate that patients with the hyper IgE syndrome have increased numbers of Fc gamma R+ PBL, most of them being B cells, whereas their T cells contain less than 0.1% Fc epsilon R+ cells.

Adolescent↗
PubMed1984

Maternal isoimmunisation resulting in combined immunodeficiency and fatal graft-versus-host disease in an infant.

Isoimmunisation to platelet and lymphocyte antigens occurred during a woman's seven pregnancies and resulted in congenital thrombocytopenia and lymphocytopenia in the last two offspring. The youngest baby died aged 16 days of severe combined immunodeficiency (SCID) with maternal graft-versus-host disease. The other affected child, however, recovered fully from thrombocytopenia and lymphocytopenia after exchange transfusion for presumed sepsis. The clinical courses indicated that an isoimmune process had caused both the thrombocytopenia and the lymphocytopenia. In accord with this conclusion, maternal titres of IgG antibodies against paternal platelets fell continuously in serial blood samples taken after the last pregnancy. Cytotoxicity studies of the maternal serum towards lymphocytes from the other siblings and from unrelated donors showed that the paternal antigen involved was a non-HLA determinant. It is postulated that isoimmune lymphocytopenia with subsequent immunological suppression is a potential mechanism for SCID and intrauterine engraftment of maternal lymphocytes.

Adult↗