Giulia C.B. Astaldi

Immunoperoxidase procedures to detect monoclonal antibodies against cell surface antigens. Quantitation of binding and staining of individual cells

An immunoperoxidase method has been developed which allows accurate and sensitive quantitation of the binding of monoclonal antibodies to cell surface antigens. Monolayers of fixed cells were prepared in wells of Terasaki micro-test plates and monoclonal antibodies bound to cell surface antigens were identified by the unlabeled antibody-enzyme method of Sternberger (1974). The cell-bound peroxidase could either be quantified per well or visualized on individual cells by the use of appropriate substrates for peroxidase. Experimental procedures are described in detail and results obtained with several monoclonal antibodies with specificity for different target cells are shown. Limitations and applications of the technique are discussed.

The in vitro effect of a thymic epithelial culture supernatant on mixed lymphocyte reactivity and intracellular cAMP levels of thymocytes and on antibody production to SRBC by nu/nu spleen cells

Abstract Addition of supernatants from rat thymic epithelial cultures (TES) to rat thymocytes stimulated with T-cell-mitogens or allogeneic cells leads to an increase in 14 C-TdR incorporation. Furthermore, in the presence of TES, spleen cells from athymic nu/nu mice exhibit an enhanced in vitro antibody production to SRBC, whereas TES has no such effect on spleen cells from T-cell-deprived mice. If TES is added together with thymocytes to T-cell-deprived spleen cell cultures, the number of plaque-forming cells to SRBC is enhanced, suggesting that TES induces a helper cell function in thymocytes which, if added alone, have no effect. TES also increases intracellular levels of cAMP in thymocytes in vitro and appears to act on a membrane site distinct from the β-adrenergic receptor. TES fails to affect mitogen responses, MLR and cAMP levels of lymphocytes from other lymphoid organs. The biological activity of TES as compared to that of thymic extracts is discussed.

A thymus-dependent serum factor induces maturation of thymocytes as evaluated by graft-versus-host reaction

Abstract Mouse thymocytes were incubated in vitro in the presence of a thymus-dependent serum factor. This factor increases the level of cellular cyclic AMP but does not affect the number of proliferating cells. After incubation with the factor, C57BL/6J thymocytes were injected into new born (C57BL/6J × C3H)F 1 hybrids. The capability of thymocytes to elicit a graft-versus-host reaction in vivo was evaluated by the spleen weight assay. Thymocytes treated with the thymic factor were found to induce a more effective graft-versus-host reaction as compared with untreated thymocytes and with thymocytes treated with inactivated thymic factor. These results are consistent with the existence of a thymocyte maturation pathway which does not involve cell proliferation and is under the influence of thymic factors.

Early events in thymocyte activation. I. Stimulation of protein synthesis by a thymus-dependent human serum factor

Abstract Human serum contains a thymus-dependent factor that raises cyclic AMP levels in thymocytes. We found that this factor stimulates protein synthesis in thymocytes cultured in vitro. This activity of serum factor is thymus-dependent, because it is absent in sera from thymectomized donors; furthermore, this effect is predominantly found on precursors of mature T cells. Incubation of thymocytes with other agents that increase cyclic AMP, induces an increase in protein synthesis similar to that observed with serum factor. Most likely, the increase in protein synthesis is one of the events following stimulation of adenylate cyclase in thymocytes that leads to cell differentiation.

Serum thymic factor in subacute sclerosing panencephalitis patients.

Abstract The activity of thymus-dependent serum factor (SF) was measured in 11 patients with subacute sclerosing panencephalitis (SSPE). A significantly decreased activity was found in 6 out of 11 patients, whereas the level of cyclic AMP in lymphocytes of the patients, a parameter for the maturation of peripheral blood lymphocytes, showed low values in 9 out of 11 patients. These findings were compared with the clinical stage of the disease and with some lymphocyte functions in vitro . No correlation was found between the SF levels and the amount of cyclic AMP in the lymphocytes or between these two parameters and the lymphocyte functions in vitro . It is concluded that a thymic humoral dysfunction is unlikely to play a role in the pathogenesis of SSPE; it appears that such a dysfunction is rather a secondary event in this disease.

Human-Mouse Hybridoma Cells Producing Antibodies

Abstract 1 Human Endothelial Culture Supernatant (HECS) has a growth-promoting activity toward hybridoma cells and increases the stability of human-mouse hybridoma cells. 2 The recovery of human-mouse hybridoma cells after fusion is strongly increased when the human lymphocytes are stimulated in vitro prior to the fusion. 3 The highest yields of hybridoma cells are obtained when the human lymphocytes are cultured with the antigen, in the presence of endothelial cells, prior to the fusion with mouse myeloma cells. 4 The recovery of stable human-mouse hybridoma cells is increased by selecting for clones producing antibodies, which can be achieved by cloning at low cell density.

THYMUS-DEPENDENT HUMAN SERUM FACTOR ACTIVE ON PRECURSORS OF MATURE T CELLS

Publisher Summary This chapter discusses the thymus-dependent human serum factor (SF) that is active on precursors of mature T cells. The chapter describes the effect of SF on cAMP level in different target cells as compared to prostaglandin (PGE1), a substance known to increase cAMP in lymphoid and nonlymphoid cells. In a study described in the chapter, SF markedly increased the level of cAMP in thymocytes, moderately in lymph node cells, and only marginally in spleen cells. Because SF does not induce any increase in the level of cAMP in mature T cells such as hydrocortisone (HC)-resistant mouse thymocytes and human peripheral blood T cells, it may be concluded that SF selectively stimulates cAMP synthesis in precursors of mature T cells. Such precursors are known to be present in small numbers among spleen cells and lymph node cells and to represent a substantial portion of HC-sensitive thymocytes.

A thymus-dependent human serum factor induces a decrease of terminal deoxynucleotidyl transferase in thymocytes

Abstract Previously, we have shown that a thymus-dependent human serum factor (SF) selectively acts on hydrocortisone-sensitive, immunologically immature thymocytes, inducing hydrocortisone resistance and immunological maturation as evaluated by graft-vs-host reaction. Human and mouse thymocytes were incubated with SF for different times and the activity of terminal deoxynucleotidyl transferase (TdT) was measured hereafter. A marked decrease of TdT activity was found in thymocytes exposed to SF for 240 min. These data indicate that the maturation of thymocytes induced by SF is accompanied by a decrease of TdT activity.

Production of Human Antibodies of Predefined Specificity in Vitro

Abstract 1. Fusion of human lymphocytes with an appropriate cell line can result in antibody-producing cultures. The recovery of such antibody-producing cultures is strongly dependent upon the treatment of the lymphocytes during culture prior to the fusion. We found that prestimulation of human lymphocytes in vitro with antigen and PWM, in the presence of human endothelial cells, leads to optimal recovery of antibody-producing cells. 2. We obtained antibody-producing cells by fusing stimulated human lymphocytes with mouse myeloma cells. Moreover, we fused human lymphocytes with an EBV-producing primate cell line. The resulting antibody-producing cells proved to be of purely human origin and, apparently, were the result of infection of the human B cells with EBV.

Reactivity of Monoclonal Anti-HLA Antibodies with Blood Platelets

Abstract Binding pattern of mouse monoclonal antibodies specific for determinants on HLA-A, -B and -C glycoproteins were studied. The reactivity of the antibodies was quantitated by an “unlabeled antibody” immunoperoxidase technique. Blood platelets from 4 donors were used as target cells. The following results were obtained: 1) Observed binding patterns of monoclonal anti-HLA-A2 and anti-HLA-B7 was in agreement with results of conventional HLA-typing of lymphocytes; 2) Binding curves, obtained at different dilutions of a monoclonal antibody against a non-polymorphic “backbone” HLA-determinant, showed little variation between platelets of the different donors; 3) A a saturating amount of antibody, binding of monoclonal anti-HLA-A2 to platelets showed a gene-dose effect: platelets of an HLA-A2 homozygous individual bound approximately twice the amount of anti-HLA-A2 antibody compared to platelets from a HLA-A2 heterozygous donor.