Grazia M. Giudizi

Rosette formation with protein A-coated erythrocytes: A method for detecting both IgG-bearing cells and another subset of human peripheral blood B lymphocytes

Abstract Human peripheral blood lymphocytes were tested for ability to form rosettes with human red blood cells (HRBC) coated with staphylococcal protein A (SpA-HRBC). Purified T lymphocytes did not form rosettes, but in T-cell-depleted suspensions the number of cells forming rosettes with SpA-HRBC was significantly greater than in unfractionated suspensions. When non-T-cell suspensions were depleted of Ig-bearing lymphocytes, cells forming rosettes with SpA-HRBC were no longer detectable. The number of cells forming rosettes with SpA-HRBC in either unfractionated or T-cell-depleted suspensions was significantly higher than the number forming rosettes with HRBC coated with anti-human γ chain immunosorbent-purified rabbit antibodies. Membrane components reacting with SpA-HRBC were not passively adsorbed on the cell surface and could be actively synthesized by lymphocytes. The SpA-reacting membrane components present on some B lymphocytes were sensitive to treatment with low concentrations of pronase, but other B cells maintained ability to form rosettes even after treatment with higher pronase concentrations. Incubation of T-cell-depleted lymphocyte suspensions with anti-human γ, and also with anti-human μ or anti-human δ chain F(ab′) 2 fragments induced significant reduction in the number of cells forming rosettes. After incubation of the same cell suspensions with a mixture of anti-γ, anti-μ and anti-δ (F(ab′) 2 fragments, virtually all the lymphocytes lost the ability to form rosettes with SpA-HRBC. These findings suggest that rosette formation with SpA-HRBC may be used as a method for detecting IgG-bearing cells but also to detect a subset of IgM- and/or IgD-bearing human B lymphocytes.

Different reactivity of activated human B cells to B-cell growth factor and interleukin 2 in the costimulation assay with anti-IgM antibody and in the preactivation assay with Staphylococcus bacteria

The two main assay systems which have been developed for the study of lymphokine-mediated human B-cell proliferation, i.e., the costimulation assay with anti-mu antibody and the preactivation assay with Staphylococcus aureus Cowan I (SAC) bacteria, were compared. Purified interleukin 2 (IL-2), obtained by the recombinant DNA technology (r-IL-2), enhanced the proliferative response of anti-mu-stimulated human B cells in the costimulation assay with anti-mu antibody and maintained the B-cell proliferation induced by preactivation with SAC bacteria. Although the majority of T-cell clones, established from normal peripheral blood T lymphocytes, showed production of both IL-2 and B-cell growth factor (BCGF) following phytohemagglutinin (PHA)-stimulation, some T-cell clones were found whose supernatants (PHA-SN), apparently free of IL-2, manifested strong BCGF activity in the costimulation assay with anti-mu antibody. However, the same clonal, IL-2-free, T-cell SN displayed no BCGF activity in the preactivation assay with SAC bacteria. When B cells were activated for 3 days with anti-mu antibody, followed by the addition of r-IL-2 or clonal T-cell SN containing BCGF for an additional 3 days, r-IL-2 showed the ability to maintain B-cell proliferation, whereas clonal SN containing BCGF had virtually no effect. These data indicate that the costimulation assay with anti-mu antibody explores the reactivity of normal human B cells to both BCGF and IL-2, whereas the preactivation assay with SAC bacteria, due to a shorter reactivity to BCGF of activated human B cells, essentially represents a probe for the study of IL-2-promoted B-cell proliferation.

A simple solid-phase radioimmunoassay for the measurement of IgG secreted in vitro by human lymphocytes.

A radioimmunoassay is described for measuring IgG, based on the ability of immunoglobulins of this class to inhibit the binding of radioiodinated staphylococcal protein A to IgG linked to a solid phase. The solid phase is represented by ox erythrocytes coated with anti-ox erythrocyte rabbit IgG, a reagent used for detecting cells equipped with receptors for the Fc fragment of IgG. By this assay the IgG secreted in vitro by human peripheral blood lymphocytes stimulated with PWM and those present in samples of very diluted human sera were measured. It was found that the assay is a very rapid, simple and reproducible procedure for the detection of IgG immunoglobulin at the nanogram level.

Protein a reactivity of IgM- and IgD-bearing lymphocytes from some patients with chronic lymphocytic leukemia☆

Abstract Most peripheral blood lymphocytes (PBL) from 7 out of 17 chronic lymphocytic leukemia (CLL) patients were able to form rosettes with human red blood cells coated with staphylococcal protein A (SpA-HRBC). PBL from 6 out of these 7 patients had surface IgM and IgD, whereas PBL from one patient had surface IgG. The SpA-reactive membrane component of PBL from some patients was sensitive to treatment with pronase, whereas in other patients it was not removed by enzymatic treatment. The SpA-reacting membrane component removed by pronase was reexpressed by most PBL after 72 hr in culture. Either unfractionated or T-cell-depleted PBL from 4 CLL patients showed significant DNA synthesis after stimulation with SpA-containing staphylococci. This proliferative response was peculiar of patients whose PBL formed rosettes with SpA-HRBC. These data suggest that there are monoclonal B lymphocytes which form rosettes with SpA-HRBC and are activated by SpA-containing staphylococci.

T-Cell Independent Activation of Human B Cells by Anti-Ig Antibodies and Protein A-Containing Staphylococci

The agents to be considered as polyclonal activators of human B cells (PBA) can be distinguished in two categories: the first category includes antibodies against B cell surface components, such as anti-immunoglobulin (Ig) antibodies (Chiorazzi et al., 1980) and the second one comprises of some micro-organisms, such as Ebstein-Barr virus (Tosato et al., 1980), Staphylococcus aureus (Forsgren et al., 1976), Haemophilus influenzae (Banck and For-sgren, 1978), etc.