Human blood B lymphocytes with receptors for sheep erythrocytes--their relevance in techniques to obtain T cells depleted of mature B cells.
After B lymphocyte depletion, blood lymphocytes (PBMC) from certain individuals will generate substantial quantities of immunoglobulin (Ig) when cultured with pokeweed mitogen (PWM). The present investigations were aimed at analysing the cell subset in these T cell preparations, that was responsible for Ig production especially since the quantity of Ig generated was disproportionate to numbers of contaminating sIg-bearing B cells. Ficoll-Hypaque PBMC from 17 individuals were subjected to overnight sheep erythrocyte rosetting. Rosetted cells were subjected to very slow (100 X g) density gradient centrifugation to isolate T rosettes (and deplete PBMC of B cells). In 6 of these 17 individuals, such enriched T cells repeatedly generated substantial quantities of plasmacytoid cells after an 8-day culture in the presence of PWM and helper factors. Mean values for plasmacytoid cells per 1000 cells recovered were as follows: T cells 168.16 +/- 96.7 SD, B cells 226.67 +/- 161.1, PBMC 225.67 +/- 78.9. In further experiments, contaminating surface immunoglobulin (sIg)-positive B cells (less than 2% sIg-positive) were removed from the T cell preparations by the "panning" method, i.e. layering T cells on plates precoated with antisera specific for human Ig (polyvalent), and lysis of B cells with a monoclonal antibody BA-1. In these 6 individuals, removal of B cells by both these techniques completely abolished generation of plasmacytoid cells, thus confirming that it is a contaminating B cell subset which is responsible for Ig production in these T cell preparations. These data indicate that in certain individuals there are B cells that separate out with T cells during the E-rosette isolation procedure. With double immunofluorescence techniques, it became apparent that 4.8 +/- 1.3% of TRITC-labeled sIgM-bearing B cells were also labeled with FITC-conjugated monoclonal antibody to the sheep erythrocyte receptor. Kuritani and Cooper have previously demonstrated that PWM-responsive B cell precursors of IgM, IgG, or IgA plasmacytoid cells lack sIgD and, hence, comprise about 10-15% of the total B cells in PBMC. Our data would indicate that in certain individuals, about half this subset forms E-rosettes, which may explain why both E-rosette separated T cells and enriched B cells make similar quantities of plasmacytoid cells.
D000818:Animals; D001402:B-Lymphocytes / Q000166:cytology* / Q000276:immunology; D002469:Cell Separation / Q000379:methods; D003165:Complement System Proteins / Q000276:immunology; D003602:Cytotoxicity, Immunologic; D004912:Erythrocytes / Q000276:immunology; D005455:Fluorescent Antibody Technique; D006801:Humans; D008213:Lymphocyte Activation; D011971:Receptors, Immunologic / Q000276:immunology*; D012397:Rosette Formation; D012756:Sheep; D013601:T-Lymphocytes / Q000166:cytology* / Q000276:immunology
D011971:Receptors, Immunologic; D003165:Complement System Proteins